Formulations for suprachoroidal administration such as formulations with aggregate formation

Abstract

Provided herein are pharmaceutical compositions for administration to a suprachoroidal space of an eye of a subject. The pharmaceutical compositions can include a recombinant adeno-associated virus (AAV) encoding a transgene. Also provided herein are methods for treating or preventing a disease in a subject by administering a therapeutically effective amount of the pharmaceutical compositions to the subject in need.

Description

PRIORITY

[0001] This application claims the benefit of priority to U.S. Ser. No. 63 / 328,252 filed Apr. 6, 2022, which is incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] This application contains a computer readable Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “12656-163-228_SEQLISTING.xml”, was created on Mar. 30, 2023 and is 49,621 bytes in size.1. BACKGROUND OF THE INVENTION

[0003] The human eye is a highly intricate and highly developed sensory organ, which is prone to a host of diseases and disorders. About 285 million people in the world are visually impaired, of whom 39 million are blind and 246 million have moderate to severe visual impairment (World Health Organization, 2012, “Global Data On Visual Impairments 2010,” Geneva: World Health Organization). Some of the leading causes of blindness are cataract (47%), glaucoma (12%), age-related macular degeneration (AMD) (9%), and diabetic retinopathy (5%) (World Health Organization, 2007, “Global Initiative For The Elimination Of Avoidable Blindness: Action Plan 2006-2011,” Geneva: World Health Organization).

[0004] Gene therapy has been employed in treating certain eye diseases (see, e.g. International Patent Application No. PCT / US2017 / 027650 (International Publication No. WO 2017 / 181021 A1)). Adeno-associated viruses (AAV) are an attractive tool for gene therapy due to properties of non-pathogenicity, broad host and cell type tropism range of infectivity, including both dividing and non-dividing cells, and ability to establish long-term transgene expression (e.g., Gonçalves, 2005, Virology Journal, 2:43).

[0005] Current methods used for ocular gene therapy (e.g., by intravitreous or subretinal administrations) are invasive and have serious setbacks, such as, increased risk of cataract, retinal detachment, and separation of photoreceptors from the retinal pigment epithelium (RPE) in the fovea. There is a significant unmet medical need for therapies that improve or eliminate the setbacks from current ocular gene therapy.

[0006] Adeno-associated virus (AAV), a member of the Parvoviridae family designated Dependovirus, is a small nonenveloped, icosahedral virus with single-stranded linear DNA genomes of approximately 4.7-kilobases (kb) to 6 kb. The properties of non-pathogenicity, broad host and cell type tropism range of infectivity, including both dividing and non-dividing cells, and ability to establish long-term transgene expression make AAV an attractive tool for gene therapy (e.g., Gonçalves, 2005, Virology Journal, 2.43).

[0007] The suprachoroidal space (SCS) is a region between the sclera and the choroid that expands upon injection of the drug solution (Habot-Wilner, 2019). The SCS space recovers to its pre-injection size as the injected solution is cleared by physiologic processes. The drug solution diffuses within SCS and is absorbed into adjacent tissues. Capillaries in the choroid are permeable to low molecular weight osmolytes. The present disclosure addresses an unmet need of providing pharmaceutical compositions that lead to longer residence time in the suprachoroidal space, and consequently improved efficacy.2. SUMMARY OF THE INVENTION

[0008] In one aspect, provided herein is a pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject, wherein the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, and wherein the pharmaceutical composition has an amount of viral vector aggregation such that when administered to an eye of a pig: a. the clearance time of the pharmaceutical composition is between about 5 days and about 15 days; and b. the thickness of the SCS at the site of injection is between about 400 μm and about 800 μm at a time within one hour of administration; and c. the circumferential spread of the pharmaceutical composition from the site of injection is about one-eighth or less of a surface of the choroid at a time within about one hour of administration.

[0009] In some embodiments, the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, and wherein the pharmaceutical composition comprises an ionic strength of at most about 200 mM prior to suprachoroidal administration.

[0010] In some embodiments, the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, and wherein the pharmaceutical composition comprises at least about 3% aggregated recombinant AAV prior to suprachoroidal administration.

[0011] In some embodiments, the clearance time after suprachoroidal administration of the pharmaceutical composition is equal to or greater than the clearance time after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition. In some embodiments, a circumferential spread after suprachoroidal administration of the pharmaceutical composition is smaller as compared to a circumferential spread after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition. In some embodiments, a thickness at a site of injection after suprachoroidal administration of the pharmaceutical composition is equal to or higher as compared to a thickness at a site of injection after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition. In some embodiments, an expression level of the transgene is detected in the eye for a longer period of time after suprachoroidal administration of the pharmaceutical composition as compared to a period of time that an expression level of the transgene is detected in the eye after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition. In some embodiments, the concentration of the transgene product in the eye after suprachoroidal administration of the pharmaceutical composition is equal to or higher as compared to the concentration of the transgene product in the eye after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition. In some embodiments, the concentration of the transgene product in the back of the eye (e.g., retina) after suprachoroidal administration of the pharmaceutical composition is equal to or higher as compared to the concentration of the transgene product in the back of the eye after suprachoroidal administration of a reference pharmaceutical composition, and / or the concentration of the transgene product in the outer layer of the eye (e.g., sclera) after suprachoroidal administration of the pharmaceutical composition is lower than the concentration of the transgene product in the outer layer of the eye after suprachoroidal administration of a reference pharmaceutical composition disclosed herein.

[0012] In some embodiments, the rate of transduction at a site of injection after suprachoroidal administration is equal to or higher as compared to the rate of transduction at a site of injection after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition.

[0013] In some embodiments, the clearance time is from the SCS or from the eye. In some embodiments, the clearance time is the time required for the pharmaceutical composition and / or the recombinant adeno-associated virus (AAV) vector to not be detectable in the SCS by any standard method. In some embodiments, the clearance time when the pharmaceutical composition and / or the recombinant adeno-associated virus (AAV) vector is present in the SCS in an amount that is at most about 2% or at most about 5% of the amount detectable by any standard method. In some embodiments, the clearance time is the amount of time required following injection for the thickness at the site of injection to decrease to about 1 nm or less, about 2 nm or less, about 5 nm or less, about 10 nm or less, about 25 nm or less, about 50 nm or less, about 100 nm or less, about 200 nm or less, or about 500 nm or less. In some embodiments, the clearance time is the amount of time required following injection for the thickness at the site of injection to decrease to about 500 nm or less, about 200 nm or less, about 100 nm or less, about 50 nm or less, about 25 nm or less, about 10 nm or less, or is undetectable. In some embodiments, the clearance time is the amount of time required following injection for the pharmaceutical composition to spread circumferentially from the site of injection to cover about one-sixteenth or more, about one-eighth or more, about one-fourth or more, about one-half or more, about three-fourths or more, or all of the circumference of the choroid of the eye.

[0014] In some embodiments, the transgene is not an anti-human vascular endothelial growth factor (anti-VEGF) antibody. In some embodiments, the recombinant AAV comprises components from one or more adeno-associated virus serotypes selected from the group consisting of AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, rAAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the recombinant AAV is AAV8. In some embodiments, wherein the recombinant AAV is AAV9. In some embodiments, the pharmaceutical composition has an ionic strength of about or at most about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM. In some embodiments, the pharmaceutical composition comprises at least about or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% aggregated recombinant AAV.

[0015] In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 40 mM. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 135 mM. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 20 mM. In some embodiments, the pharmaceutical composition has an average recombinant AAV diameter of about or at least about: 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, or about or at least about 100 nm (e.g., as measured with dynamic light scattering).

[0016] In some embodiments, the pharmaceutical composition has an average recombinant AAV diameter that is at least 2 times higher, at least 3 times higher, at least 4 times higher, at least 5 times higher, at least 6 times higher, at least 7 times higher, at least 8 times higher, at least 9 times higher, at least 10 times higher, at least 15 times higher, at least 20 times higher, at least 50 times higher, at least 100 times higher, at least 5% higher, at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40%, at least 45% higher, at least 50% higher, at least 55% higher, at least 60% higher, at least 65% higher, at least 70% higher, at least 75% higher, at least 80% higher, at least 85% higher, at least 90% higher, at least 95% higher, at least 100% higher, at least 150% higher, or at least 200% higher, at least 250% higher, or at least 300%, at least 400% higher, or at least 500% higher than an average recombinant AAV diameter in the reference pharmaceutical composition. In some embodiments, the circumferential spread after suprachoroidal administration of the pharmaceutical composition is smaller by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%. In some embodiments, the clearance time after suprachoroidal administration of the pharmaceutical composition is greater by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500%.

[0017] In some embodiments, the clearance time after suprachoroidal administration of the pharmaceutical composition is of about 6 days to about 15 days, about 7 days to about 15 days, about 8 days to about 15 days, about 9 days to about 15 days, about 10 days to about 15 days, about 11 days to about 15 days, about 12 days to about 15 days, about 13 days to about 15 days, about 14 days to about 15 days, about 5 days to about 14 days, about 5 days to about 13 days, about 5 days to about 12 days, about 5 days to about 11 days, about 5 days to about 10 days, about 5 days to about 9 days, about 5 days to about 8 days, about 5 days to about 7 days, or about 5 days to about 6 days. In some embodiments, the clearance time after suprachoroidal administration of the pharmaceutical composition is not prior to about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days. In some embodiments, the clearance time after suprachoroidal administration of the reference pharmaceutical composition is of at most about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0018] In some embodiments, the clearance time after suprachoroidal administration of the pharmaceutical composition is of about 30 minutes to about 20 hours, about 2 hours to about 20 hours, about 30 minutes to about 24 hours, about 1 hour to about 2 hours, about 30 minutes to about 90 days, about 30 minutes to about 60 days, about 30 minutes to about 30 days, about 30 minutes to about 21 days, about 30 minutes to about 14 days, about 30 minutes to about 7 days, about 30 minutes to about 3 days, about 30 minutes to about 2 days, about 30 minutes to about 1 day, about 4 hours to about 90 days, about 4 hours to about 60 days, about 4 hours to about 30 days, about 4 hours to about 21 days, about 4 hours to about 14 days, about 4 hours to about 7 days, about 4 hours to about 3 days, about 4 hours to about 2 days, about 4 hours to about 1 day, about 4 hours to about 8 hours, about 4 hours to about 16 hours, about 4 hours to about 20 hours, about 1 day to about 90 days, about 1 day to about 60 days, about 1 day to about 30 days, about 1 day to about 21 days, about 1 day to about 14 days, about 1 day to about 7 days, about 1 day to about 3 days, about 2 days to about 90 days, about 3 days to about 90 days, about 3 days to about 60 days, about 3 days to about 30 days, about 3 days to about 21 days, about 3 days to about 14 days, or about 3 days to about 7 days.

[0019] In some embodiments, the clearance time after suprachoroidal administration of the pharmaceutical composition is not prior to about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days. In some embodiments, the clearance time after suprachoroidal administration of the reference pharmaceutical composition is of at most about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the clearance time is from the SCS or from the eye. In some embodiments, the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is higher by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%.

[0020] In some embodiments, the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is about 500 μm to about 3.0 mm, 750 μm to about 2.8 mm, about 750 μm to about 2.5 mm, about 750 μm to about 2 mm, or about 1 mm to about 2 mm. In some embodiments, the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is of at least about 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm. In some embodiments, the thickness at the site of injection after suprachoroidal administration of the reference pharmaceutical composition is of at most about 1 nm, 5 nm, 10 nm, 25 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm.

[0021] In some embodiments, the thickness of the SCS at the site of injection is about 400 μm to about 700 μm, about 400 μm to about 600 μm, about 400 μm to about 500 μm, about 500 μm to about 800 μm, about 600 μm to about 800 μm, 700 μm to about 800 μm at a time within one hour of administration. In some embodiments, the time within one hour of administration is within about 5 minutes of administration, within about 10 minutes of administration, within about 15 minutes of administration, within about 20 minutes of administration, within about 30 minutes of administration, within about 45 minutes of administration, or within about 60 minutes of administration.

[0022] In some embodiments, the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition persists for at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours, at least eighteen hours, at least twenty-four hours, at least two days, at least three days, at least five days, at least ten days, at least twenty-one days, at least one month, at least six weeks, at least two months, at least three months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least one year, at least three years, or at least five years.

[0023] In some embodiments, the circumferential spread of the pharmaceutical composition from the site of injection is about one-eighth or less of a surface of the choroid at a time within about 5 minutes of administration, within about 10 minutes of administration, within about 15 minutes of administration, within about 20 minutes of administration, within about 30 minutes of administration, within about 45 minutes of administration, or within about 60 minutes of administration. In some embodiments, the circumferential spread from the site of injection is about one-sixteenth or less of a surface of the choroid.

[0024] In some embodiments, the concentration of the transgene product in the eye after suprachoroidal administration of the pharmaceutical composition is higher by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%.

[0025] In some embodiments, the longer period of time after suprachoroidal administration of the pharmaceutical composition is longer by at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days. In some embodiments, the transgene is detected in the eye after suprachoroidal administration of the pharmaceutical composition for at least about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days. In some embodiments, the transgene is detected in the eye after suprachoroidal administration of the reference pharmaceutical composition for at most about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, or 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days after.

[0026] In some embodiments, the rate of transduction at the site of injection after suprachoroidal administration of the pharmaceutical composition is higher by at least about 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%.

[0027] In some embodiments, the recombinant AAV stability in the pharmaceutical composition is at least about 50% of the recombinant AAV stability in the reference pharmaceutical composition. In some embodiments, the recombinant AAV stability is determined by infectivity of the recombinant AAV. In some embodiments, the recombinant AAV stability is determined by a level of free DNA released by the recombinant AAV. In some embodiments, the pharmaceutical composition comprises at least about 50% more, about 25% more, about 15% more, about 10% more, about 5% more, about 4% more, about 3% more, about 2% more, about 1% more, about 0% more, about 1% less, about 2% less, about 5% less, about 7% less, about 10% less, about 2 times more, about 3 times more, about 2 times less, about 3 times less free DNA as compared to a level of free DNA in the reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has an infectivity that is about 50% lower, about the same, or at least about 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher as compared to the infectivity of the recombinant AAV in the reference pharmaceutical composition.

[0028] In some embodiments, the transgene is a transgene suitable to treat, or otherwise ameliorate, prevent or slow the progression of a disease of interest. In some embodiments, the human subject is diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), or Batten disease. In other embodiments, the human subject is diagnosed with glaucoma or non-infectious uveitis. In some embodiments, the human subject is diagnosed with kallikrein-related disease.

[0029] In some embodiments, the AAV encodes Palmitoyl-Protein Thioesterase 1 (PPT1) or Tripeptidyl-Peptidase 1 (TPP1). In other embodiments, the AAV encodes anti-kallikrein antibody or antigen-binding fragment, anti-TNF fusion protein, anti-TNF antibody or antigen-binding fragment, anti-C3 antibody or antigen-binding fragment, or anti-C5 antibody or antigen-binding fragment.

[0030] In some embodiments, the amount of the recombinant AAV genome copies is based on a vector genome concentration. In some embodiments, the amount of the recombinant AAV genome copies is based on genome copies per administration. In some embodiments, the amount of the recombinant AAV genome copies is based on total genome copies administered to the human subject. In some embodiments, the genome copies per administration is the genome copies of the recombinant AAV per suprachoroidal administration. In some embodiments, the total genome copies administered is the total genome copies of the recombinant AAV administered suprachoroidally.

[0031] In some embodiments, the vector genome concentration (VGC) is of about 3×109 GC / mL, about 1×1010 GC / mL, about 1.2×1010 GC / mL, about 1.6×1010 GC / mL, about 4×1010 GC / mL, about 6×1010 GC / mL, about 2×1011 GC / mL, about 2.4×1011 GC / mL, about 2.5×1011 GC / mL, about 3×1011 GC / mL, about 6.2×1011 GC / mL, about 1×1012 GC / mL, about 2.5×1012 GC / mL, about 3×1012 GC / mL, about 5×1012 GC / mL, about 1.5×1013 GC / mL, about 2×1013 GC / mL, or about 3×1013 GC / mL. In some embodiments, the total genome copies administered is about 6.0×1010 genome copies, about 1.6×1011 genome copies, about 2.5×1011 genome copies, about 5.0×1011 genome copies, about 1.5×1012 genome copies, about 3×1012 genome copies, about 1.0×1012 genome copies, about 2.5×1012 genome copies, or about 3.0×1013 genome copies. In some embodiments, the genome copies per administration is about 6.0×1010 genome copies, about 1.6×1011 genome copies, about 2.5×1011 genome copies, about 5.0×1011 genome copies, about 3×1012 genome copies, about 1.0×1012 genome copies, about 1.5×1012 genome copies, about 2.5×1012 genome copies, or about 3.0×1013 genome copies.

[0032] In some embodiments, the pharmaceutical composition is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, twenty-five times, or thirty times. In some embodiments, the reference pharmaceutical composition is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, twenty-five times, or thirty times. In some embodiments, the pharmaceutical composition is administered once in one day, twice in one day, three times in one day, four times in one day, five times in one day, six times in one day, or seven times in one day. In some embodiments, the reference pharmaceutical composition is administered once in one day, twice in one day, three times in one day, four times in one day, five times in one day, six times in one day, or seven times in one day. In some embodiments, the reference pharmaceutical composition comprises DPBS and sucrose.

[0033] In one aspect, provided herein is a method of preparing a pharmaceutical composition comprising: (i) preparing a composition comprising phosphate-buffered saline, sucrose, and a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene; and (ii) admixing a solution comprising phosphate-buffered saline and sucrose to the composition, wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the composition.

[0034] In one aspect, provided herein is a method of preparing a pharmaceutical composition comprising admixing a solution comprising phosphate-buffered saline and sucrose to a composition, wherein the composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the composition.

[0035] In one aspect, provided herein is a kit comprising: (i) a composition comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene; and (ii) a solution comprising phosphate-buffered saline and sucrose. In some embodiments, the kit further comprises instructions for admixing the composition with the solution. In some embodiments, the instructions comprise instructions on admixing the solution with the composition to obtain a pharmaceutical composition.

[0036] In some embodiments, the composition comprises a phosphate-buffered saline and sucrose. In some embodiments, the composition comprises 4% sucrose. In some embodiments, the solution comprises 10% sucrose. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 135 mM. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 40 mM. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 20 mM. In some embodiments, the pharmaceutical composition has substantially the same tonicity or osmolality as the composition. In some embodiments, at least some of the aggregated recombinant AAV in the pharmaceutical composition disaggregate after the pharmaceutical composition is administered to the suprachoroidal space of an eye of a human subject. In some embodiments, aggregation of the recombinant AAV is reversed to unaggregated AAV or to monomers upon suprachoroidal administration of the pharmaceutical composition. In some embodiments, the composition comprises potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and optionally a surfactant. In some embodiments, the composition comprises modified Dulbecco's phosphate-buffered saline solution, and optionally a surfactant. In some embodiments, the composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, and a surfactant. In some embodiments, the solution comprises a phosphate-buffered sodium chloride and sucrose. In some embodiments, the solution comprises 10% Sucrose, 2.70 mM potassium chloride, 8.10 mM sodium phosphate dibasic anhydrous, 1.47 mM potassium phosphate monobasic, 292 mM sucrose, 0.001% (0.01 mg / mL) poloxamer 188, pH 7.4, and wherein the composition and solution are admixed at a composition to solution ratio of 1 to 9.

[0037] In some embodiments, admixing the solution with the composition dilutes the composition by about or at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold. In some embodiments, admixing the solution with the composition occurs on the same day that the pharmaceutical composition is administered to the suprachoroidal space of an eye of a human subject. In some embodiments, admixing the solution with the composition occurs within 24 hours of the pharmaceutical composition being administered to the suprachoroidal space of an eye of a human subject.

[0038] In some embodiments, the pharmaceutical composition is stored prior to administration to a human subject. In some embodiments, the pharmaceutical composition is stored at about room temperature, 20° C., 4° C., or −80° C. In some embodiments, the recombinant AAV comprises components from AAV8 and the pharmaceutical composition has an ionic strength between about 30 mM to about 60 mM. In some embodiments, the recombinant AAV comprises components from AAV8 and the pharmaceutical composition has an ionic strength between about 30 mM to about 50 mM. In some embodiments, the recombinant AAV comprises components from AAV8 and the pharmaceutical composition has an ionic strength between about 20 mM to about 60 mM. In some embodiments, the recombinant AAV comprises components from AAV8 and the pharmaceutical composition has an ionic strength of about, at most about, or at least about: 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, or higher than 70 mM. In some embodiments, the average particle diameter of the AAV is between about, at least about, or at most about: 15-70 nm, 20-60 nm, 25 nm-55 nm, 30-50 nm, or 30-70 nm (e.g., as measured by DLS). In some embodiments, the recombinant AAV comprises components from AAV9 and the pharmaceutical composition has an ionic strength between about 15 mM to about 30 mM. In some embodiments, the recombinant AAV comprises components from AAV9 and the pharmaceutical composition has an ionic strength between about 10 mM to about 50 mM. In some embodiments, the recombinant AAV comprises components from AAV9 and the pharmaceutical composition has an ionic strength between about 10 mM to about 35 mM. In some embodiments, the recombinant AAV comprises components from AAV9 and the pharmaceutical composition has an ionic strength of about, at most about, or at least about: 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, or higher than 70 mM. In some embodiments, the recombinant AAV comprises components from AAV2 and the pharmaceutical composition has an ionic strength between about 100 mM to about 200 mM. In some embodiments, the pharmaceutical composition comprises modified Dulbecco's phosphate-buffered saline solution, and optionally a surfactant. In some embodiments, the pharmaceutical composition comprises potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and optionally a surfactant. In some embodiments, the pharmaceutical composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, and optionally a surfactant. In some embodiments, the pharmaceutical composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, 0.001% (0.01 mg / mL) poloxamer 188, pH 7.4.

[0039] In one aspect provided herein is a pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject, wherein the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, wherein the pharmaceutical composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, 0.001% (0.01 mg / mL) poloxamer 188, pH 7.4. In some embodiments, the pharmaceutical composition comprises a lower amount of AAV empty capsids as compared to a reference pharmaceutical composition. In some embodiments, the amount of the AAV empty capsids in the pharmaceutical composition is lower by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the amount of the AAV empty capsids in the reference pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises an ionic strength of about or at most about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, or 60 mM. In some embodiments, the reference pharmaceutical composition comprises an ionic strength of more than about 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, or more than about 200 mM.

[0040] In one aspect provided herein is a method of reducing or eliminating AAV empty capsids in a pharmaceutical composition, the method comprising: a. introducing a solution comprising an ionic strength of at most about 60 mM to a formulation comprising a mixture of a recombinant adeno-associated virus (AAV) vector and AAV empty capsids; and b. removing at least some of the AAV empty capsids from the formulation, wherein the formulation after step b is prepared into a pharmaceutical composition comprising the recombinant adeno-associated virus (AAV) vector, wherein the recombinant AAV vector comprises an expression cassette encoding a transgene, and wherein the pharmaceutical composition is suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject. In some embodiments, the method further comprises introducing another solution comprising an ionic strength of at least about 80 mM to the formulation after step b. In some embodiments, a pharmaceutical composition is produced by the method.

[0041] In one aspect provided herein is a method of reducing or eliminating AAV empty particles in a population of AAV particles, wherein the population of AAV particles comprises empty AAV particles and AAV particles comprising an expression cassette encoding a transgene, and wherein the method comprises: a. incubating the population of AAV particles in a solution at an ionic strength of at most about 60 mM, thereby creating aggregates of the AAV particles comprising the expression cassette encoding the transgene; and b. removing at least a portion of the empty AAV particles from the population of AAV particles. In some embodiments, the method further comprises incubating the population of AAV particles after step b with another solution comprising an ionic strength of at least about 80 mM. In some embodiments, a pharmaceutical composition comprising the population of AAV particles is obtained after step b of the method.

[0042] In some embodiments, the amount of the AAV empty capsids in the formulation is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99%, or 100% after step b. as compared to the amount of the AAV empty capsids in the formulation prior to step a. In some embodiments, the amount of the empty AAV particles in the population of AAV particles is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99%, or 100% after step b. as compared to the amount of the empty AAV particles in the population of AAV particles prior to step a.

[0043] In some embodiments, the solution comprises an ionic strength of about 30 to about 50 mM. In some embodiments, the solution comprises an ionic strength of about 15 to 50 mM. In some embodiments, the solution comprises an ionic strength of at most about 50 mM. In some embodiments, the another solution comprises an ionic strength of about or at least about 150 mM.2.1 ILLUSTRATIVE EMBODIMENTS

[0044] 1. A pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject, wherein the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, and wherein the pharmaceutical composition has an amount of viral vector aggregation such that when administered to an eye of a pig:

[0045] a. the clearance time of the pharmaceutical composition is between about 5 days and about 15 days; and

[0046] b. the thickness of the SCS at the site of injection is between about 400 μm and about 800 μm at a time within one hour of administration; and

[0047] c. the circumferential spread of the pharmaceutical composition from the site of injection is about one-eighth or less of a surface of the choroid at a time within about one hour of administration.

[0048] 2. The pharmaceutical composition of paragraph 1, wherein the pharmaceutical composition comprises an ionic strength of at most about 200 mM prior to suprachoroidal administration.

[0049] 3. The pharmaceutical composition of paragraph 1, wherein the pharmaceutical composition comprises at least about 3% aggregated recombinant AAV prior to suprachoroidal administration.

[0050] 4. The pharmaceutical composition of any one of paragraphs 1-3, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is equal to or greater than the clearance time after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition.

[0051] 5. The pharmaceutical composition of any one of paragraphs 1-3, wherein a circumferential spread after suprachoroidal administration of the pharmaceutical composition is smaller as compared to a circumferential spread after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition.

[0052] 6. The pharmaceutical composition of any one of paragraphs 1-3, wherein a thickness at a site of injection after suprachoroidal administration of the pharmaceutical composition is equal to or higher as compared to a thickness at a site of injection after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition.

[0053] 7. The pharmaceutical composition of any one of paragraphs 1-3, wherein an expression level of the transgene is detected in the eye for a longer period of time after suprachoroidal administration of the pharmaceutical composition as compared to a period of time that an expression level of the transgene is detected in the eye after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition.

[0054] 8. The pharmaceutical composition of any one of paragraphs 1-3, wherein the concentration of the transgene product in the eye after suprachoroidal administration of the pharmaceutical composition is equal to or higher as compared to the concentration of the transgene product in the eye after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition;

[0055] optionally wherein the concentration of the transgene product in the back of the eye (e.g., retina) after suprachoroidal administration of the pharmaceutical composition is equal to or higher as compared to the concentration of the transgene product in the back of the eye after suprachoroidal administration of a reference pharmaceutical composition, and / or the concentration of the transgene product in the outer layer of the eye (e.g., sclera) after suprachoroidal administration of the pharmaceutical composition is lower than the concentration of the transgene product in the outer layer of the eye after suprachoroidal administration of a reference pharmaceutical composition.

[0056] 9. The pharmaceutical composition of any one of paragraphs 1-3, wherein the rate of transduction at a site of injection after suprachoroidal administration is equal to or higher as compared to the rate of transduction at a site of injection after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition.

[0057] 10. The pharmaceutical composition of any one of paragraphs 2-9, wherein the transgene is not an anti-human vascular endothelial growth factor (anti-VEGF) antibody.

[0058] 11. The pharmaceutical composition of any one of paragraphs 2-10, wherein the recombinant AAV comprises components from one or more adeno-associated virus serotypes selected from the group consisting of AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, rAAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

[0059] 12. The pharmaceutical composition of any one of paragraphs 1-11, wherein the recombinant AAV is AAV8.

[0060] 13. The pharmaceutical composition of any one of paragraphs 1-11, wherein the recombinant AAV is AAV9.

[0061] 14. The pharmaceutical composition of any one of paragraphs 1-13, wherein the pharmaceutical composition has an ionic strength of about or at most about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM.

[0062] 15. The pharmaceutical composition of any one of paragraphs 1-14, wherein the pharmaceutical composition comprises at least about or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% aggregated recombinant AAV.

[0063] 16. The pharmaceutical composition of any one of paragraphs 1-15, wherein the pharmaceutical composition has an ionic strength of about or of at most about 40 mM.

[0064] 17. The pharmaceutical composition of any one of paragraphs 1-15, wherein the pharmaceutical composition has an ionic strength of about or of at most about 135 mM.

[0065] 18. The pharmaceutical composition of any one of paragraphs 1-15, wherein the pharmaceutical composition has an ionic strength of about or of at most about 20 mM.

[0066] 19. The pharmaceutical composition of any one of paragraphs 1-18, wherein the pharmaceutical composition has an average recombinant AAV diameter of about or at least about: 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, or about or at least about 100 nm as measured by dynamic light scattering.

[0067] 20. The pharmaceutical composition of any one of paragraphs 4-19, wherein the pharmaceutical composition has an average recombinant AAV diameter that is at least 2 times higher, at least 3 times higher, at least 4 times higher, at least 5 times higher, at least 6 times higher, at least 7 times higher, at least 8 times higher, at least 9 times higher, at least 10 times higher, at least 15 times higher, at least 20 times higher, at least 50 times higher, at least 100 times higher, at least 5% higher, at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40%, at least 45% higher, at least 50% higher, at least 55% higher, at least 60% higher, at least 65% higher, at least 70% higher, at least 75% higher, at least 80% higher, at least 85% higher, at least 90% higher, at least 95% higher, at least 100% higher, at least 150% higher, or at least 200% higher, at least 250% higher, or at least 300%, at least 400% higher, or at least 500% higher than an average recombinant AAV diameter in the reference pharmaceutical composition.

[0068] 21. The pharmaceutical composition of any one of paragraphs 5 and 10-20, wherein the circumferential spread after suprachoroidal administration of the pharmaceutical composition is smaller by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%.

[0069] 22. The pharmaceutical composition of any one of paragraphs 4 and 10-21, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is greater by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500%.

[0070] 23. The pharmaceutical composition of any one of paragraphs 1-22, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is of about 6 days to about 15 days, about 7 days to about 15 days, about 8 days to about 15 days, about 9 days to about 15 days, about 10 days to about 15 days, about 11 days to about 15 days, about 12 days to about 15 days, about 13 days to about 15 days, about 14 days to about 15 days, about 5 days to about 14 days, about 5 days to about 13 days, about 5 days to about 12 days, about 5 days to about 11 days, about 5 days to about 10 days, about 5 days to about 9 days, about 5 days to about 8 days, about 5 days to about 7 days, or about 5 days to about 6 days.

[0071] 24. The pharmaceutical composition of any one of paragraphs 1-23, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is not prior to about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

[0072] 25. The pharmaceutical composition of any one of paragraphs 4-24, wherein the clearance time after suprachoroidal administration of the reference pharmaceutical composition is of at most about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0073] 26. The pharmaceutical composition of any one of paragraphs 1-25, wherein the clearance time is from the SCS or from the eye.

[0074] 27. The pharmaceutical composition of any one of paragraphs 1-26, wherein the clearance time is the time required for the pharmaceutical composition and / or the recombinant adeno-associated virus (AAV) vector to not be detectable in the SCS by any standard method.

[0075] 28. The pharmaceutical composition of any one of paragraphs 1-26, wherein the clearance time when the pharmaceutical composition and / or the recombinant adeno-associated virus (AAV) vector is present in the SCS in an amount that is at most about 2% or at most about 5% of the amount detectable by any standard method.

[0076] 29. The pharmaceutical composition of any one of paragraphs 1-26, wherein the clearance time is the amount of time required following injection for the thickness at the site of injection to decrease to about 500 nm or less, about 200 nm or less, about 100 nm or less, about 50 nm or less, about 25 nm or less, about 10 nm or less, or is undetectable.

[0077] 30. The pharmaceutical composition of any one of paragraphs 1-26, wherein the clearance time is the amount of time required following injection for the pharmaceutical composition to spread circumferentially from the site of injection to cover about one-sixteenth or more, about one-eighth or more, about one-fourth or more, about one-half or more, about three-fourths or more, or all of the circumference of the choroid of the eye.

[0078] 31. The pharmaceutical composition of any one of paragraphs 6 and 10-30, wherein the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is higher by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%.

[0079] 32. The pharmaceutical composition of any one of paragraphs 1-31, wherein the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is about 500 μm to about 3.0 mm, 750 μm to about 2.8 mm, about 750 μm to about 2.5 mm, about 750 μm to about 2 mm, or about 1 mm to about 2 mm.

[0080] 33. The pharmaceutical composition of any one of paragraphs 1-32, wherein the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is of at least about 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, or 700 μm, 800 μm, 900 μm, 1000 μm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm.

[0081] 34. The pharmaceutical composition of any one of paragraphs 6 and 10-33, wherein the thickness at the site of injection after suprachoroidal administration of the reference pharmaceutical composition is of at most about 1 nm, 5 nm, 10 nm, 25 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm.

[0082] 35. The pharmaceutical composition of any one of paragraphs 1-34, wherein the thickness of the SCS at the site of injection is about 400 μm to about 700 μm, about 400 μm to about 600 μm, about 400 μm to about 500 μm, about 500 μm to about 800 μm, about 600 μm to about 800 μm, 700 μm to about 800 μm at a time within one hour of administration.

[0083] 36. The pharmaceutical composition of paragraph 35, wherein the time within one hour of administration is within about 5 minutes of administration, within about 10 minutes of administration, within about 15 minutes of administration, within about 20 minutes of administration, within about 30 minutes of administration, within about 45 minutes of administration, or within about 60 minutes of administration.

[0084] 37. The pharmaceutical composition of any one of paragraphs 1-36, wherein the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition persists for at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours, at least eighteen hours, at least twenty-four hours, at least two days, at least three days, at least five days, at least ten days, at least twenty-one days, at least one month, at least six weeks, at least two months, at least three months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least one year, at least three years, or at least five years.

[0085] 38. The pharmaceutical composition of any one of paragraphs 1-36, wherein the circumferential spread of the pharmaceutical composition from the site of injection is about one-eighth or less of a surface of the choroid at a time within about 5 minutes of administration, within about 10 minutes of administration, within about 15 minutes of administration, within about 20 minutes of administration, within about 30 minutes of administration, within about 45 minutes of administration, or within about 60 minutes of administration.

[0086] 39. The pharmaceutical composition of paragraph 38, wherein the circumferential spread from the site of injection is about one-sixteenth or less of a surface of the choroid.

[0087] 40. The pharmaceutical composition of any one of paragraphs 8 and 10-39, wherein the concentration of the transgene product in the eye after suprachoroidal administration of the pharmaceutical composition is higher by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%.

[0088] 41. The pharmaceutical composition of any one of paragraphs 7 and 10-40, wherein the longer period of time after suprachoroidal administration of the pharmaceutical composition is longer by at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days.

[0089] 42. The pharmaceutical composition of any one of paragraphs 1-41, wherein the transgene is detected in the eye after suprachoroidal administration of the pharmaceutical composition for at least about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days.

[0090] 43. The pharmaceutical composition of any one of paragraphs 4-42, wherein the transgene is detected in the eye after suprachoroidal administration of the reference pharmaceutical composition for at most about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, or 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days after.

[0091] 44. The pharmaceutical composition of any one of paragraphs 9 and 10-43, wherein the rate of transduction at the site of injection after suprachoroidal administration of the pharmaceutical composition is higher by at least about 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%.

[0092] 45. The pharmaceutical composition of any one of paragraphs 2-44, wherein the recombinant AAV stability in the pharmaceutical composition is at least about 50% of the recombinant AAV stability in the reference pharmaceutical composition.

[0093] 46. The pharmaceutical composition of paragraph 45, wherein the recombinant AAV stability is determined by infectivity of the recombinant AAV.

[0094] 47. The pharmaceutical composition of paragraph 45, wherein the recombinant AAV stability is determined by a level of free DNA released by the recombinant AAV.

[0095] 48. The pharmaceutical composition of paragraph 47, wherein the pharmaceutical composition comprises at least about 50% more, about 25% more, about 15% more, about 10% more, about 5% more, about 4% more, about 3% more, about 2% more, about 1% more, about 0% more, about 1% less, about 2% less, about 5% less, about 7% less, about 10% less, about 2 times more, about 3 times more, about 2 times less, about 3 times less free DNA as compared to a level of free DNA in the reference pharmaceutical composition.

[0096] 49. The pharmaceutical composition of paragraph 46, wherein the recombinant AAV in the pharmaceutical composition has an infectivity that is about 50% lower, about the same, or at least about 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher as compared to the infectivity of the recombinant AAV in the reference pharmaceutical composition.

[0097] 50. The pharmaceutical composition of any one of paragraphs 1-49, wherein the transgene is a transgene suitable to treat, or otherwise ameliorate, prevent or slow the progression of a disease of interest.

[0098] 51. The pharmaceutical composition of any one of paragraphs 1-50, wherein the human subject is diagnosed with nAMD (wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), or Batten disease.

[0099] 52. The pharmaceutical composition of any one of paragraphs 1-50, wherein the human subject is diagnosed with glaucoma, non-infectious uveitis, or kallikrein-related disease.

[0100] 53. The pharmaceutical composition of any one of paragraphs 1-3, 4-9, and 10-52, wherein the AAV encodes Palmitoyl-Protein Thioesterase 1 (PPT1), Tripeptidyl-Peptidase 1 (TPP1), anti-TNF fusion protein, anti-kallikrein antibody or antigen-binding fragment, anti-TNF antibody or antigen-binding fragment, anti-C3 antibody or antigen-binding fragment, or anti-C5 antibody or antigen-binding fragment.

[0101] 54. The pharmaceutical composition of any one of paragraphs 4-53, wherein the amount of the recombinant AAV genome copies is based on a vector genome concentration.

[0102] 55. The pharmaceutical composition of any one of paragraphs 4-53, wherein the amount of the recombinant AAV genome copies is based on genome copies per administration.

[0103] 56. The pharmaceutical composition of any one of paragraphs 4-53, wherein the amount of the recombinant AAV genome copies is based on total genome copies administered to the human subject.

[0104] 57. The pharmaceutical composition of paragraph 55, wherein the genome copies per administration is the genome copies of the recombinant AAV per suprachoroidal administration.

[0105] 58. The pharmaceutical composition of paragraph 56, wherein the total genome copies administered is the total genome copies of the recombinant AAV administered suprachoroidally.

[0106] 59. The pharmaceutical composition of paragraph 54, wherein the vector genome concentration (VGC) is of about 3×109 GC / mL, about 1×1010 GC / mL, about 1.2×1010 GC / mL, about 1.6×1010 GC / mL, about 4×1010 GC / mL, about 6×1010 GC / mL, about 2×1011 GC / mL, about 2.4×1011 GC / mL, about 2.5×1011 GC / mL, about 3×1011 GC / mL, about 6.2×1011 GC / mL, about 1×1012 GC / mL, about 2.5×1012 GC / mL, about 3×1012 GC / mL, about 5×1012 GC / mL, about 6×1012 GC / mL, about 1.5×1013 GC / mL, about 2×1013 GC / mL, or about 3×1013 GC / mL.

[0107] 60. The pharmaceutical composition of any one of paragraphs 56 and 58, wherein the total number of genome copies administered is about 6.0×1010 genome copies, about 1.6×1011 genome copies, about 2.5×1011 genome copies, about 3×1011 genome copies, about 5.0×1011 genome copies, about 6×1011 genome copies, about 3×1012 genome copies, about 1.0×1012 genome copies, about 1.5×1012 genome copies, about 2.5×1012 genome copies, or about 3.0×1013 genome copies.

[0108] 61. The pharmaceutical composition of any one of paragraphs 55 and 57, wherein the total number of genome copies per administration is about 6.0×1010 genome copies, about 1.6×1011 genome copies, about 2.5×1011 genome copies, about 3×1011 genome copies, about 5.0×1011 genome copies, about 6×1011 genome copies, about 3×1012 genome copies, about 1.0×1012 genome copies, about 1.5×1012 genome copies, about 2.5×1012 genome copies, or about 3.0×1013 genome copies.

[0109] 62. The pharmaceutical composition of any one of paragraphs 2-61, wherein the pharmaceutical composition is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, twenty five times, or thirty times.

[0110] 63. The pharmaceutical composition of any one of paragraphs 4-62, wherein the reference pharmaceutical composition is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, twenty-five times, or thirty times.

[0111] 64. The pharmaceutical composition of any one of paragraphs 2-63, wherein the pharmaceutical composition is administered once in one day, twice in one day, three times in one day, four times in one day, five times in one day, six times in one day, or seven times in one day.

[0112] 65. The pharmaceutical composition of any one of paragraphs 4-63, wherein the reference pharmaceutical composition is administered once in one day, twice in one day, three times in one day, four times in one day, five times in one day, six times in one day, or seven times in one day.

[0113] 66. The pharmaceutical composition of any one of paragraphs 2-65, wherein the reference pharmaceutical composition comprises DPBS and sucrose.

[0114] 67. A method of preparing a pharmaceutical composition comprising:

[0115] a. preparing a composition comprising phosphate-buffered saline, sucrose, and a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene; and

[0116] b. admixing a solution comprising phosphate-buffered saline and sucrose to the composition,wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the composition.

[0117] 68. A method of preparing a pharmaceutical composition comprising admixing a solution comprising phosphate-buffered saline and sucrose to a composition, wherein the composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the composition.

[0118] 69. A kit comprising:

[0119] a. a composition comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene; and

[0120] b. a solution comprising phosphate-buffered saline and sucrose.

[0121] 70. The kit of paragraph 69, wherein the kit further comprises instructions for admixing the composition with the solution.

[0122] 71. The method of paragraph 68 or the kit of paragraph 69, wherein the composition comprises a phosphate-buffered saline and sucrose.

[0123] 72. The method of any one of paragraphs 67 and 71, or the kit of any one of paragraphs 69 and 71, wherein the composition comprises 4% sucrose.

[0124] 73. The method of any one of paragraphs 67, 68, 71 and 72, or the kit of any one of paragraphs 69-72, wherein the solution comprises 10% sucrose.

[0125] 74. The method of any one of paragraphs 67, 68, and 71-73, or the pharmaceutical composition of any one of paragraphs 1-66, wherein the pharmaceutical composition has an ionic strength of about or of at most about 135 mM.

[0126] 75. The method of any one of paragraphs 67, 68, and 71-74, or the pharmaceutical composition of any one of paragraphs 1-66, wherein the pharmaceutical composition has an ionic strength of about or of at most about 40 mM.

[0127] 76. The method of any one of paragraphs 67, 68, and 71-75, or the pharmaceutical composition of any one of paragraphs 1-66, wherein the pharmaceutical composition has an ionic strength of about or of at most about 20 mM.

[0128] 77. The method of any one of paragraphs 67, 68, and 71-76, or the pharmaceutical composition of any one of paragraphs 1-66, wherein the pharmaceutical composition has substantially the same tonicity or osmolality as the composition.

[0129] 78. The method of any one of paragraphs 67, 68, and 71-77, or the pharmaceutical composition of any one of paragraphs 1-66, wherein at least some of the aggregated recombinant AAV in the pharmaceutical composition disaggregate after the pharmaceutical composition is administered to the suprachoroidal space of an eye of a human subject.

[0130] 79. The method of any one of paragraphs 67, 68, and 71-78, or the pharmaceutical composition of any one of paragraphs 1-66, wherein aggregation of the recombinant AAV is reversed to unaggregated AAV or to monomers upon suprachoroidal administration of the pharmaceutical composition.

[0131] 80. The method of any one of paragraphs 67, 68, and 71-79, or the kit of any one of paragraphs 69-72, wherein the composition comprises potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and optionally a surfactant.

[0132] 81. The method of any one of paragraphs 67, 68, and 71-80, or the kit of any one of paragraphs 69-72 and 80, wherein the composition comprises modified Dulbecco's phosphate-buffered saline solution, and optionally a surfactant.

[0133] 82. The method of any one of paragraphs 67, 68, and 71-81, or the kit of any one of paragraphs 69-72 and 80-81, wherein the composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, and a surfactant.

[0134] 83. The method of any one of paragraphs 67, 68, and 71-82, or the kit of any one of paragraphs 69-72 and 80-82, wherein the solution comprises a phosphate-buffered sodium chloride and sucrose.

[0135] 84. The kit of paragraph 70, wherein the instructions comprise instructions on admixing the solution with the composition to obtain a pharmaceutical composition.

[0136] 85. The method of any one of paragraphs 67, 68, and 71-83, or the kit of paragraph 84, wherein the admixing the solution with the composition dilutes the composition by about or at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold.

[0137] 86. The method of any one of paragraphs 67, 68, 71-83 and 85, or the kit of any one of paragraphs 84-85, wherein the admixing the solution with the composition occurs on the same day that the pharmaceutical composition is administered to the suprachoroidal space of an eye of a human subject.

[0138] 87. The method of any one of paragraphs 67, 68, 71-83 and 85-86, or the kit of any one of paragraphs 84-86, wherein the admixing the solution with the composition occurs within 24 hours of the pharmaceutical composition being administered to the suprachoroidal space of an eye of a human subject.

[0139] 88. The method of any one of paragraphs 67, 68, 71-83 and 85-87, or the kit of any one of paragraphs 84-87, or the pharmaceutical composition of any one of paragraphs 2-66 and 74-79, wherein the pharmaceutical composition is stored prior to administration to a human subject.

[0140] 89. The method of any one of paragraphs 67, 68, 71-83 and 85-88, or the kit of any one of paragraphs 84-88, or the pharmaceutical composition of any one of paragraphs 2-66, 74-79, and 88, wherein the pharmaceutical composition is stored at about room temperature, 20° C., 4° C., or −80° C.

[0141] 90. The method of any one of paragraphs 67, 68, 71-83 and 85-89, or the kit of any one of paragraphs 84-89, or the pharmaceutical composition of any one of paragraphs 2-66, 74-79, and 88-89, wherein the pharmaceutical composition comprises about 1.0×1012 to about 3.0×1012 genome copies of the recombinant AAV.

[0142] 91. The method of any one of paragraphs 67, 68, 71-83 and 85-90, or the kit of any one of paragraphs 84-90, wherein the recombinant AAV comprises components from one or more adeno-associated virus serotypes selected from AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, rAAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

[0143] 92. The method of any one of paragraphs 67, 68, 71-83 and 85-91, or the kit of any one of paragraphs 84-91, or the pharmaceutical composition of any one of paragraphs 2-66, 74-79, and 88-90, wherein the recombinant AAV comprises components from AAV8 and the pharmaceutical composition has an ionic strength between about 30 mM to about 60 mM.

[0144] 93. The method of any one of paragraphs 67, 68, 71-83 and 85-91, or the kit of any one of paragraphs 84-91, or the pharmaceutical composition of any one of paragraphs 2-66, 74-79, and 88-90, wherein the recombinant AAV comprises components from AAV9 and the pharmaceutical composition has an ionic strength between about 15 mM to about 30 mM.

[0145] 94. The method of any one of paragraphs 67, 68, 71-83 and 85-91, or the kit of any one of paragraphs 84-91, or the pharmaceutical composition of any one of paragraphs 1-66, 74-79, and 88-90, wherein the recombinant AAV comprises components from AAV2 and the pharmaceutical composition has an ionic strength between about 100 mM to about 200 mM.

[0146] 95. The pharmaceutical composition of any one of paragraphs 1-66, 74-79, and 88-94, wherein the pharmaceutical composition comprises modified Dulbecco's phosphate-buffered saline solution, and optionally a surfactant.

[0147] 96. The pharmaceutical composition of any one of paragraphs 1-66, 74-79, and 88-95, wherein the pharmaceutical composition comprises potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and optionally a surfactant.

[0148] 97. The pharmaceutical composition of any one of paragraphs 1-66, 74-79, and 88-96, wherein the pharmaceutical composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, and optionally a surfactant.

[0149] 98. The pharmaceutical composition of any one of paragraphs 1-66, 74-79, and 88-96, wherein the pharmaceutical composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, 0.001% (0.01 mg / mL) poloxamer 188, pH 7.4.

[0150] 99. A pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject, wherein the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, wherein the pharmaceutical composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, 0.001% (0.01 mg / mL) poloxamer 188, pH 7.4.

[0151] 100. The pharmaceutical composition of any one of paragraphs 1-66, 74-79, and 88-99, wherein the pharmaceutical composition comprises a lower amount of AAV empty capsids as compared to a reference pharmaceutical composition.

[0152] 101. The pharmaceutical composition of paragraph 100, wherein the amount of the AAV empty capsids in the pharmaceutical composition is lower by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the amount of the AAV empty capsids in the reference pharmaceutical composition.

[0153] 102. The pharmaceutical composition of any one of paragraphs 1-66, 74-79, and 88-101, wherein the pharmaceutical composition comprises an ionic strength of about or at most about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, or 60 mM.

[0154] 103. The pharmaceutical composition of any one of paragraphs 100-102, wherein the reference pharmaceutical composition comprises an ionic strength of more than about 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, or more than about 200 mM.

[0155] 104. A method of reducing or eliminating AAV empty capsids in a pharmaceutical composition, the method comprising:

[0156] a. introducing a solution comprising an ionic strength of at most about 60 mM to a formulation comprising a mixture of a recombinant adeno-associated virus (AAV) vector and AAV empty capsids; and

[0157] b. removing at least some of the AAV empty capsids from the formulation, wherein the formulation after step b is prepared into a pharmaceutical composition comprising the recombinant adeno-associated virus (AAV) vector, wherein the recombinant AAV vector comprises an expression cassette encoding a transgene, and wherein the pharmaceutical composition is suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject.

[0158] 105. A method of reducing or eliminating AAV empty particles in a population of AAV particles, wherein the population of AAV particles comprises empty AAV particles and AAV particles comprising an expression cassette encoding a transgene, and wherein the method comprises:

[0159] a. incubating the population of AAV particles in a solution at an ionic strength of at most about 60 mM, thereby creating aggregates of the AAV particles comprising the expression cassette encoding the transgene; and

[0160] b. removing at least a portion of the empty AAV particles from the population of AAV particles.

[0161] 106. The method of paragraph 104, wherein the method further comprises introducing another solution comprising an ionic strength of at least about 80 mM to the formulation after step b.

[0162] 107. The method of paragraph 105, wherein the method further comprises incubating the population of AAV particles after step b with another solution comprising an ionic strength of at least about 80 mM.

[0163] 108. The method of paragraph 104 or 106, wherein the amount of the AAV empty capsids in the formulation is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99%, or 100% after step b. as compared to the amount of the AAV empty capsids in the formulation prior to step a.

[0164] 109. The method of paragraph 105 or 107, wherein the amount of the empty AAV particles in the population of AAV particles is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99%, or 100% after step b. as compared to the amount of the empty AAV particles in the population of AAV particles prior to step a.

[0165] 110. The method of any one of paragraphs 104-109, wherein the solution comprises an ionic strength of about 30 to about 50 mM.

[0166] 111. The method of any one of paragraphs 104-109, wherein the solution comprises an ionic strength of about 15 to 50 mM.

[0167] 112. The method of any one of paragraphs 104-109, wherein the solution comprises an ionic strength of at most about 50 mM.

[0168] 113. The method of any one of paragraphs 104-109, wherein the another solution comprises an ionic strength of about or at least about 150 mM.

[0169] 114. A pharmaceutical composition produced by the method of any one of paragraphs 104, 106, and 108-113.

[0170] 115. A pharmaceutical composition comprising the population of AAV particles obtained after step b of the method of any one of paragraphs 105 and 107-113.

[0171] 116. The method or the kit of paragraph 81 or 82, wherein the solution comprises 10% Sucrose, 2.70 mM potassium chloride, 8.10 mM sodium phosphate dibasic anhydrous, 1.47 mM potassium phosphate monobasic, 292 mM sucrose, 0.001% (0.01 mg / mL) poloxamer 188, pH 7.4, and wherein the composition and solution are admixed at a composition to solution ratio of 1 to 9.3. BRIEF DESCRIPTION OF THE DRAWINGS

[0172] FIG. 1. Overview of induced clustering of AAV capsids using a low salt and / or low ionic strength diluent.

[0173] FIG. 2. Graph showing impact of ionic strength and salt concentration on diameter of AAV.

[0174] FIGS. 3A-3B. Electron microscopy visual representation of the impact of ionic strength and salt concentration on diameter of AAV. FIG. 3A shows AAVs in a control or reference pharmaceutical composition. FIG. 3A shows that AAVs do not aggregate in a reference pharmaceutical composition. FIG. 3B shows AAV aggregation in low ionic strength solutions (pharmaceutical compositions comprising lower ionic strength as compared to the reference pharmaceutical composition).

[0175] FIG. 4. Graph showing average apparent diameter of AAV clusters in solutions containing different ionic strengths and salt amounts as measured from the time of dilution to about 21 hours post dilution, at 25° C. The control comprises recombinant AAV in modified DPBS with 4% sucrose. The two-times, four-times, and eight-times dilution comprises recombinant AAV in modified DPBS with 4% sucrose diluted with different amounts of phosphate-buffered 10% sucrose diluent to obtain the two-times, four-times, and eight-times dilutions. Clusters were stable at 25° C. for at least 21 hours. At around 21.8 hours a spike of sodium chloride was added to obtain solutions with at least 75 mM salt level, which reversed the clusters back to monomers.

[0176] FIG. 5. Graph showing cumulants dynamic light scattering (DLS) intensity-weighted average apparent diameter for recombinant AAV. FIG. 5 shows the initial induced clustering data from FIG. 4 up to about 21.8 hours of AAV in the control, in the two-times, four-times, and eight-times dilutions into low ionic strength phosphate-buffered 10% sucrose diluent (refer to FIG. 4).

[0177] FIG. 6. Graph showing cumulants dynamic light scattering (DLS) intensity-weighted average apparent diameter for recombinant AAV. FIG. 6 shows the reversal of the induced clustering, in the two-times, four-times, and eight-times dilutions into low ionic strength phosphate-buffered 10% sucrose diluent. Reversal of induced clustering was effected with a spike of salt at about 21.8 hours of AAV (refer to FIG. 4).

[0178] FIG. 7. Graph showing cumulants diameter of recombinant AAV in modified DPBS with 4% sucrose formulation, in an induced-clustering low-salt solution (a ten-times dilution) prepared by dilution with phosphate-buffered 10% sucrose diluent, and after reversal of the clustering with addition of salt. The figure also shows that clustering remained after the samples were heated to 37° C.

[0179] FIGS. 8A-8B. Impact of ionic strength on AAV8 in F3 and AAV9 in F4 colloidal stability. FIG. 8A: AAV8 empty capsids (circle), AAV8 lot 1 (square). FIG. 8B: AAV9 empty capsids (circle), AAV9 lot 1 (square).

[0180] FIG. 9. Illustration of a representative purification of AAV (e.g. with HF-TFF) based on the colloidal stability difference between empty and full capsids under low ionic strength conditions.

[0181] FIG. 10. UV absorption comparison between DNA, empty capsid, and AAV full capsid, utilized to assess empty vs. full capsids in a composition.4. DETAILED DESCRIPTION OF THE INVENTION

[0182] Provided herein are pharmaceutical compositions comprising recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene suitable for administration to a suprachoroidal space (SCS) of an eye of a subject. The subject can be a subject diagnosed with one of more diseases described in Section 4.5. The AAV vectors are described in Section 4.4 and dosages of such vectors are described in Section 4.3. In some embodiments, pharmaceutical compositions provided in Section 4.1 are formulated such that they have one or more functional properties described in Section 4.2. In certain embodiments, the pharmaceutical composition provided herein has various advantages, for example, increased or slower clearance time (Section 4.2.1); decreased circumferential spread (Section 4.2.2); increased SCS thickness (Section 4.2.3); increased AAV level and rate of transduction (rate of infection) at the site of injection (Section 4.2.4); and increased concentration of the transgene after the pharmaceutical composition is administered in the SCS. Without being bound by theory, the functional properties can be achieved using compositions comprising aggregated viral vectors formulations as disclosed in Section 4.1. Also provided herein are assays that may be used in related studies (Section 4.6).4.1 Formulation of Pharmaceutical Composition

[0183] The disclosure provides a pharmaceutical composition suitable for suprachoroidal administration comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene. In some embodiments, several pharmaceutical compositions (e.g., diluted formulation or lower ionic strength formulation) having different aggregation levels of AAV are used to administer an AAV encoding a transgene.

[0184] In some embodiments, the pharmaceutical composition has a higher level of AAV aggregation than a comparable pharmaceutical composition (a reference pharmaceutical composition). In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition comprise a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same vector genome concentration. In some embodiments, the pharmaceutical composition and a reference pharmaceutical composition have the same amount of genome copies. In some embodiments, the pharmaceutical composition and a reference pharmaceutical composition are each administered to a subject using the same amount of genome copies. In some embodiments, the pharmaceutical composition has a percentage of aggregated viral vectors that is higher than the percentage of viral vector aggregation of a control. In some embodiments, the pharmaceutical composition has a percentage of aggregated viral vectors that is higher than the percentage of aggregated viral vectors of a solution normally used for subretinal injection. In some embodiments, the reference pharmaceutical composition has less viral vector aggregation than the pharmaceutical composition. In some embodiments, the reference pharmaceutical composition has the same or similar percentage of viral vector aggregation than the pharmaceutical composition. In some embodiments, the reference pharmaceutical composition is a control solution (e.g., DPBS, PBS, water, or HBSS). In some embodiments, the reference pharmaceutical composition comprises the AAV in a control solution (e.g., DPBS, PBS, water, or HBSS). In some embodiments, the reference pharmaceutical composition comprises sucrose. In some embodiments, the reference pharmaceutical composition is a pharmaceutical composition commonly used for AAV subretinal injection.

[0185] In some embodiments, the pharmaceutical composition comprising the AAV is diluted so that the AAV in the pharmaceutical composition forms clustering or aggregation of AAV. In some embodiments, the pharmaceutical composition is diluted with any solution suitable to provide AAV solutions containing lower ionic strength and salt content. In some embodiments, the pharmaceutical composition is diluted with any solution suitable to reduce the ionic strength of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is diluted with a solution having reduced ionic excipient sodium chloride to induce AAV clustering. In some embodiments, the pharmaceutical composition is diluted with a solution containing reduced ionic excipient and / or increased non-ionic excipient. In some embodiments, the pharmaceutical composition is diluted with a solution containing reduced ionic excipient sodium chloride and increased non-ionic excipient sucrose. In some embodiments, the pharmaceutical composition is diluted with phosphate-buffered 10% sucrose solutions. In some embodiments, the pharmaceutical composition is diluted with solutions comprising varying non-ionic excipient levels (e.g., 4% sucrose, 6% sucrose, 8% sucrose, 10% sucrose, 15% sucrose, or 20% sucrose). In some embodiments, the pharmaceutical composition is diluted with a solution comprising 4% sucrose, 6% sucrose, or 10% sucrose. In some embodiments, the pharmaceutical composition is diluted with solutions comprising varying ionic excipient levels (e.g., a solution containing reduced sodium chloride concentration as compared to the sodium chloride concentration in the pharmaceutical composition). In some embodiments, the pharmaceutical composition has the same tonicity / osmolality before and after dilution. In some embodiments, the pharmaceutical composition has the same tonicity / osmolality as the reference pharmaceutical composition or a control. In some embodiments, the pharmaceutical composition has a higher tonicity / osmolality than a reference pharmaceutical composition or a control. In some embodiments, the pharmaceutical composition has a lower tonicity / osmolality than a reference pharmaceutical composition or a control. In some embodiments, the pharmaceutical composition has a tonicity / osmolality equal to or greater than 240 mOsm / kg. In some embodiments, the pharmaceutical composition or the reference pharmaceutical composition has a tonicity / osmolality that is at least 240 mOsm / kg.

[0186] In some embodiments, the pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) is diluted before administration (e.g., suprachoroidal administration). In some embodiments, the pharmaceutical composition is diluted on the same day as the administration (e.g., suprachoroidal administration). In some embodiments, the pharmaceutical composition is diluted about 20 hours or about 24 hours before the administration (e.g., suprachoroidal administration). In some embodiments, the pharmaceutical composition is diluted about 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, two weeks, three weeks, four weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, three years, four years, five years, ten years, fifteen years, twenty years (or longer) before administration. In some embodiments, the pharmaceutical composition is diluted at most about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, two weeks, three weeks, four weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, three years, four years, five years, ten years, fifteen years, twenty years (or longer) before administration.

[0187] In some embodiments, the pharmaceutical composition is diluted and kept at room temperature (e.g., after dilution or prior to administration). In some embodiments, the pharmaceutical composition is diluted and kept at about 20° C. after the dilution. In some embodiments, the pharmaceutical composition is diluted and kept at about 25° C. after the dilution. In some embodiments, the pharmaceutical composition is diluted and kept at 4° C. after the dilution. In some embodiments, the pharmaceutical composition is diluted and kept at −20° C. after the dilution. In some embodiments, the pharmaceutical composition is diluted and kept at −80° C. after the dilution. In some embodiments, the pharmaceutical composition is diluted and is flash frozen after the dilution. In some embodiments, the undiluted pharmaceutical composition, the reference pharmaceutical composition, the diluted pharmaceutical composition, and / or the diluted reference pharmaceutical composition are suitable for long-term storage (e.g., at an appropriate temperature). In some embodiments, long-term storage is about or at least about 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, or longer than 5 years.

[0188] In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has an ionic strength (or contains an ionic excipient concentration) that is about or at most about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 255 mM, 260 mM, 265 mM, 270 mM, 275 mM, 280 mM, 285 mM, 290 mM, 295 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 800 mM, 900 mM, or at most about 1000 mM. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has an ionic strength (or contains an ionic excipient concentration) that is about or at most about 15 mM, 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 130 mM, 150 mM, 175 mM, 200 mM, or 300 mM. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has an ionic strength (or contains an ionic excipient concentration) in the range of about 5 mM to about 140 mM, of about 15 mM to about 150 mM, of about 5 mM to about 65 mM, of about 5 mM to about 200 mM, of about 15 mM to about 134 mM, of about 10 mM to about 200 mM, of about 20 mM to about 45 mM, of about 15 mM to about 300 mM, of about 5 mM to about 600 mM, of about 15 mM to about 600 mM, of about 10 mM to about 550 mM, or of about 15 mM to about 70 mM. In some embodiments, or the diluted reference pharmaceutical composition has an ionic strength (or contains an ionic excipient concentration) that is at most about 40 mM. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has an ionic strength (or contains an ionic excipient concentration) that is at most about 20 mM. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has an ionic strength (or contains an ionic excipient concentration) that is at most about 100 mM. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has an ionic strength (or contains an ionic excipient concentration) that is at most about 200 mM. In some embodiments, the reference pharmaceutical composition or the undiluted pharmaceutical composition has an ionic strength that is at least about or about 80 mM, 100 mM, 120 mM, 150 mM, 200 mM, or higher than 200 mM.

[0189] In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has a lower ionic strength than the undiluted pharmaceutical composition prior to dilution. In some embodiments, the pharmaceutical composition is diluted about or at most about two-times, three-times, four-times, five-times, six-times, seven-times, eight-times, nine-times, ten-times, fifteen-times, twenty-times, thirty-times, forty-times, fifty-times, or one hundred-times. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has an ionic strength that is below the ionic strength necessary to induce clustering of a viral vector (e.g., clustering threshold). In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has an ionic strength that is below the ionic strength for AAV clustering. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has an ionic strength that is two-times below the ionic strength for AAV clustering. In some embodiments, the clustering threshold for a viral vector is determined. In some embodiments, the clustering threshold is determined by any suitable method or any suitable assay disclosed in Section 4.6. In some embodiments, the clustering threshold for AAV8 correlates an ionic strength of about 60 mM. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an ionic strength that is about half the ionic strength for clustering threshold (e.g., 30 mM). In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an ionic strength that is about or at most about 30 mM. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an ionic strength that is about two-thirds the ionic strength for clustering threshold (e.g., 40 mM). In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an ionic strength that is about or at most about 40 mM. In some embodiments, the clustering threshold for AAV9 correlates to an ionic strength of about 30 mM. In some embodiments, the clustering threshold for AAV2 correlates to an ionic strength of about 200 mM. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an ionic strength that is about half the ionic strength for clustering threshold (e.g., 15 mM or 100 mM). In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an ionic strength that is about two-thirds the ionic strength for clustering threshold (e.g., 20 mM or 134 mM). In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an ionic strength that is about or at most about 20 mM. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an ionic strength that is about or at most about 134 mM. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an ionic strength that is about three-fourths the ionic strength for clustering threshold. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an ionic strength that is about two-fifths the ionic strength for clustering threshold.

[0190] In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has an ionic strength that is about or at most 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% the ionic strength for clustering threshold.

[0191] In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has a salt concentration (e.g., sodium chloride) that is about or at most about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 255 mM, 260 mM, 265 mM, 270 mM, 275 mM, 280 mM, 285 mM, 290 mM, 295 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 800 mM, 900 mM, or at most about 1000 mM. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has a salt concentration (e.g., sodium chloride) that is about or at most about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 200 mM, or 300 mM. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has a salt concentration (e.g., sodium chloride) that is about or at most about 10 mM, 20 mM, 40 mM, 60 mM, 100 mM, or 150 mM. In some embodiments, the reference pharmaceutical composition or an undiluted pharmaceutical composition comprises a salt (e.g., sodium chloride) concentration of at about or at least about 80 mM, 100 mM, 120 mM 150 mM, 175 mM, 200 mM or higher.

[0192] In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has a tonicity / osmolality that is at least about 240 mOsm / kg. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has a tonicity / osmolality that is at least about 100 mOsm / kg, 150 mOsm / kg, 160 mOsm / kg, 170 mOsm / kg, 180 mOsm / kg, 190 mOsm / kg, 200 mOsm / kg, 210 mOsm / kg, 220 mOsm / kg, 230 mOsm / kg, 240 mOsm / kg, 250 mOsm / kg, 260 mOsm / kg, 270 mOsm / kg, 280 mOsm / kg, 290 mOsm / kg, 300 mOsm / kg, 310 mOsm / kg, 320 mOsm / kg, 340 mOsm / kg, 350 mOsm / kg, 360 mOsm / kg, 370 mOsm / kg, 380 mOsm / kg, 390 mOsm / kg, 400 mOsm / kg, 450 mOsm / kg, 500 mOsm / kg, 550 mOsm / kg, 600 mOsm / kg, 650 mOsm / kg, or 700 mOsm / kg. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has a tonicity / osmolality that is at least about 240 mOsm / kg to about 600 mOsm / kg, 240 mOsm / kg to about 350 mOsm / kg, at least about 220 mOsm / kg to about 400 mOsm / kg, or at least about 200 mOsm / kg to about 500 mOsm / kg. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition has a tonicity / osmolality that is at between about 240 mOsm / kg to about 600 mOsm / kg.

[0193] In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition comprises at least some aggregated viral vectors. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition comprises about or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% aggregated viral vectors (e.g., aggregated AAV). In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition comprises from about 1% to about 20%, 1% to about 10%, 1% to about 50%, 3% to about 95%, 3% to about 90%, 3% to about 80%, 3% to about 70%, 3% to about 60%, 3% to about 50%, 3% to about 40%, 3% to about 30%, 3% to about 20%, 3% to about 15%, 3% to about 10%, 5% to about 95%, 5% to about 90%, 5% to about 80%, 5% to about 70%, 5% to about 60%, 5% to about 50%, 5% to about 40%, 5% to about 30%, 5% to about 20%, 5% to about 15%, 5% to about 10%, 10% to about 95%, 10% to about 90%, 10% to about 80%, 10% to about 70%, 10% to about 60%, 10% to about 50%, 10% to about 40%, 10% to about 30%, 10% to about 20%, 10% to about 15%, 15% to about 95%, 15% to about 90%, 15% to about 80%, 15% to about 70%, 15% to about 60%, 15% to about 50%, 15% to about 40%, 15% to about 30%, 15% to about 20%, 20% to about 95%, 20% to about 90%, 20% to about 80%, 20% to about 70%, 20% to about 60%, 20% to about 50%, 20% to about 40%, 20% to about 30%, 30% to about 95%, 30% to about 90%, 30% to about 80%, 30% to about 70%, 30% to about 60%, 30% to about 50%, 30% to about 40%, 40% to about 95%, 40% to about 90%, 40% to about 80%, 40% to about 70%, 40% to about 60%, or 40% to about 50% aggregated viral vectors (e.g., aggregated AAV).

[0194] In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition comprises at least 2 times more, at least 3 times more, at least 4 times more, at least 5 times more, at least 6 times more, at least 7 times more, at least 8 times more, at least 9 times more, at least 10 times more, at least 15 times more, at least 20 times more, at least 50 times more, at least 100 times more, at least 5% more, at least 10% more, at least 15% more, at least 20% more, at least 25% more, at least 30% more, at least 35% more, at least 40%, at least 45% more, at least 50% more, at least 55% more, at least 60% more, at least 65% more, at least 70% more, at least 75% more, at least 80% more, at least 85% more, at least 90% more, at least 95% more, at least 100% more, at least 150% more, or at least 200% more, at least 250% more, or at least 300%, at least 400% more, or at least 500% more aggregated viral vectors (e.g., aggregated AAV) as compared to a control solution or to the pharmaceutical composition prior to dilution or to the reference pharmaceutical composition prior to dilution.

[0195] In some embodiments, a molecular diameter of viral vectors (e.g., level of viral vector aggregation) is determined by any suitable method or any suitable assay (see Section 4.6). In some embodiments, a molecular diameter of viral vectors is measured to determine the amount of viral vector aggregation (e.g., AAV aggregation). In some embodiments, the molecular diameter of the viral vectors in the pharmaceutical composition (e.g., diluted pharmaceutical composition) or in the diluted reference pharmaceutical composition higher than the molecular diameter of the viral vectors in the pharmaceutical composition prior to dilution, or in a control solution, or in the reference pharmaceutical composition prior to dilution. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) comprises viral vectors that have (e.g., in average) a diameter that is at least 2 times higher, at least 3 times higher, at least 4 times higher, at least 5 times higher, at least 6 times higher, at least 7 times higher, at least 8 times higher, at least 9 times higher, at least 10 times higher, at least 15 times higher, at least 20 times higher, at least 50 times higher, at least 100 times higher, at least 5% higher, at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40%, at least 45% higher, at least 50% higher, at least 55% higher, at least 60% higher, at least 65% higher, at least 70% higher, at least 75% higher, at least 80% higher, at least 85% higher, at least 90% higher, at least 95% higher, at least 100% higher, at least 150% higher, or at least 200% higher, at least 250% higher, or at least 300%, at least 400% higher, or at least 500% higher than the diameter of viral vectors in a control solution or in the pharmaceutical composition prior to dilution or in the reference pharmaceutical composition. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) or the diluted reference pharmaceutical composition comprises viral vectors that have (e.g., in average) diameter that is about or at least about 10 nm, 15 nm, 20 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 450 nm, 500 nm, or over 500 nm. In some embodiments, the pharmaceutical composition prior to dilution, or the reference pharmaceutical composition prior to dilution, or a control comprises viral vectors that have (e.g., in average) diameter that is about or at most about 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, or 100 nm. In some embodiments, the pharmaceutical composition prior to dilution, or the reference pharmaceutical composition prior to dilution, or a control comprises viral vectors that have (e.g., in average) diameter that is about or at most 25 nm, 30 nm, 35 nm, or 40 nm.

[0196] In some embodiments, molecular diameter or level of viral vector aggregation is measured 30 minutes, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, one day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, two weeks, three weeks, four weeks, 2 months, 3 months, 4 months, 5 months 6 months, 1 year, 2 years 5 years, or more than 5 years after the pharmaceutical composition is diluted. In some embodiments, molecular diameter or level of viral vector aggregation is measured prior to administration (e.g., suprachoroidal administration). In some embodiments, molecular diameter or level of viral vector aggregation is measured right before administration (e.g., suprachoroidal administration). In some embodiments, molecular diameter or level of viral vector aggregation is measured 30 minutes, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, or 2 days before administration of the pharmaceutical composition (e.g., diluted pharmaceutical composition), the diluted reference pharmaceutical composition, the undiluted pharmaceutical composition, undiluted reference pharmaceutical composition, or of a control (e.g., suprachoroidal administration). In some embodiments, molecular diameter or level of viral vector aggregation is measured after long-term storage (e.g., storage at 25° C., 20° C., 4° C., −80° C.). In some embodiments, molecular diameter or level of viral vector aggregation is measured after a flash frozen diluted pharmaceutical composition is thawed.

[0197] In some embodiments, the diluted pharmaceutical composition, the undiluted pharmaceutical composition, a reference pharmaceutical composition, or a control have the same vector genome concentration. In some embodiments, the diluted pharmaceutical composition, the undiluted pharmaceutical composition, a reference pharmaceutical composition, or a control have the same amount of genome copies. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has at least the same vector genome concentration as the undiluted pharmaceutical composition (or as a control or a reference pharmaceutical composition). In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has at least the same amount of genome copies as the undiluted pharmaceutical composition (or as a control or a reference pharmaceutical composition). In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has at least the same viral vector potency (e.g., AAV in vitro potency) as the undiluted pharmaceutical composition (or as a control or a reference pharmaceutical composition).

[0198] In some embodiments, the ionic strength of the pharmaceutical composition (e.g., diluted pharmaceutical composition) is increased after administration (e.g., suprachoroidal administration). In some embodiments, the level of viral vector aggregation (clustering) is reduced after administration. In some embodiments, viral vector aggregation (clustering) in a pharmaceutical composition prior to administration allows for the viral vector or active ingredient to remain at the site of injection longer as compared to a solution comprising the viral vector but with no viral vector aggregation or as compared to a solution comprising the viral vector having a reduced amount of viral vector aggregation. In some embodiments, viral vector aggregation (clustering) in a pharmaceutical composition prior to administration results in increased thickness at the site of injection after administration as compared to the thickness at the site of injection obtained after a solution comprising the viral vector but with no viral vector aggregation or after a solution comprising the viral vector having a reduced amount of viral vector aggregation is administered. In some embodiments, viral vector aggregation (clustering) in a pharmaceutical composition prior to administration results in a smaller circumferential spread at the site of injection after administration as compared to the circumferential spread at the site of injection obtained after a solution comprising the viral vector but with no viral vector aggregation or after a solution comprising the viral vector having a reduced amount of viral vector aggregation is administered. In some embodiments, viral vector aggregation (clustering) in a pharmaceutical composition prior to administration results in a higher clearance time at the site of injection after administration as compared to the clearance time at the site of injection obtained after a solution comprising the viral vector but with no viral vector aggregation or after a solution comprising the viral vector having a reduced amount of viral vector aggregation is administered. In some embodiments, viral vector aggregation (clustering) in a pharmaceutical composition prior to administration results in a reduced level of vasodilation and / or vascular leakage after administration as compared to the level of vasodilation and / or vascular leakage obtained after a solution comprising the viral vector but with no viral vector aggregation or after a solution comprising the viral vector having a reduced amount of viral vector aggregation is administered. In some embodiments, viral vector aggregation (clustering) in a pharmaceutical composition prior to administration results in a higher rate of transduction (rate of infection) at the site of injection after administration as compared to the rate of transduction (rate of infection) at the site of injection obtained after a solution comprising the viral vector but with no viral vector aggregation or after a solution comprising the viral vector having a reduced amount of viral vector aggregation is administered. In some embodiments, viral vector aggregation (clustering) in a pharmaceutical composition prior to administration results in a higher AAV levels at the site of injection after administration as compared to the AAV levels at the site of injection obtained after a solution comprising the viral vector but with no viral vector aggregation or after a solution comprising the viral vector having a reduced amount of viral vector aggregation is administered. In some embodiments, viral vector aggregation (clustering) in a pharmaceutical composition prior to administration results in a higher transgene expression levels in the eye after administration as compared to the transgene expression levels in the eye obtained after a solution comprising the viral vector but with no viral vector aggregation or after a solution comprising the viral vector having a reduced amount of viral vector aggregation is administered. In some embodiments, a diluted pharmaceutical composition refers to a pharmaceutical composition having a lower ionic strength and / or a lower salt concentration as compared to a reference pharmaceutical composition. In some embodiments, a diluted pharmaceutical composition refers to a pharmaceutical composition comprising an ionic strength of at most about 200 mM. In some embodiments, a diluted pharmaceutical composition refers to a pharmaceutical composition comprising at least about 3% viral vector aggregation (e.g., AAV aggregation).

[0199] In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation sufficient to expand at least a portion of the site of injection (e.g. SCS) to a thickness of at least 500 μm or about 500 μm to about 3 mm, for at least two hours after administration. In some embodiments, the amount of viral vector aggregation (e.g., AAV aggregation) of the pharmaceutical composition (e.g., diluted pharmaceutical composition) is sufficient to expand the site of injection (e.g. SCS) to a thickness of about 750 μm to about 2.8 mm, about 750 μm to about 2.5 mm, about 750 μm to about 2 mm, or about 1 mm to about 2 mm. In some embodiments, the amount of viral vector aggregation (e.g., AAV aggregation) in the pharmaceutical composition (e.g., diluted pharmaceutical composition) is sufficient to expand the site of injection (e.g. SCS) to a thickness of about 500 μm to about 3.0 mm for at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours, at least eighteen hours, at least twenty-four hours, at least two days, at least three days, at least five days, at least ten days, at least twenty-one days, at least one month, at least six weeks, at least two months, at least three months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least one year, at least three years, or at least five years after the administration. In some embodiments, the amount of viral vector aggregation (e.g., AAV aggregation) in the pharmaceutical composition (e.g., diluted pharmaceutical composition) is sufficient to expand the site of injection (e.g. SCS) to a thickness of about 1 mm to about 3 mm for at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours, at least eighteen hours, or at least twenty-four hours after administration. In some embodiments, the amount of viral vector aggregation (e.g., AAV aggregation) in the pharmaceutical composition (e.g., diluted pharmaceutical composition) is sufficient to expand the site of injection (e.g. SCS) to a thickness of about 1 mm to about 2 mm for at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours, at least eighteen hours, at least twenty-four hours, at least two days, at least three days, at least five days, at least ten days, at least twenty-one days, at least one month, at least six weeks, at least two months, at least three months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least one year, at least three years, or at least five years after the administration. In some embodiments, the amount of viral vector aggregation (e.g., AAV aggregation) in the pharmaceutical composition (e.g., diluted pharmaceutical composition) is sufficient to expand the site of injection (e.g. SCS) to a thickness of about 2 mm to about 3 mm for at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours, at least eighteen hours, at least twenty-four hours, at least two days, at least three days, at least five days, at least ten days, at least twenty-one days, at least one month, at least six weeks, at least two months, at least three months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least one year, at least three years, or at least five years after the administration. In some embodiments, the amount of viral vector aggregation (e.g., AAV aggregation) in the pharmaceutical composition (e.g., diluted pharmaceutical composition) is sufficient to expand the site of injection (e.g. SCS) to a thickness of about 750 μm to about 2.8 mm, about 750 μm to about 2.5 mm, about 750 μm to about 2 mm, or about 1 mm to about 2 mm for an indefinite period. An indefinite period may be achieved due, at least in part, to the stability of the pharmaceutical composition (e.g., diluted pharmaceutical composition) in the site of injection (e.g. SCS).

[0200] In some embodiments, a pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation (e.g., AAV aggregation) sufficient to expand the site of injection (e.g. SCS) to a thickness of at least 500 μm, or about 500 μm to about 3 mm. In some embodiments, a pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation (e.g., AAV aggregation) sufficient to expand the site of injection (e.g. SCS) to a thickness of at least about 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, or larger than 10 mm. In some embodiments, a reference pharmaceutical composition or an undiluted pharmaceutical composition is capable to expand the site of injection to a thickness of at most about 1 nm, 5 nm, 10 nm, 25 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm.

[0201] Also provided herein are methods of treating a disease (e.g., an ocular disease) described in Section 4.5 using the pharmaceutical compositions (e.g., diluted pharmaceutical composition) disclosed herein. In some embodiments, a method of treating an ocular disease includes administering an effective amount of the pharmaceutical composition (e.g., recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene) to a subject (e.g., human). In some embodiments, the pharmaceutical composition is administered in the suprachoroidal space (SCS) of an eye of the subject. In some embodiments, the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response when administered to the SCS is less than the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response when administered subretinally. In some embodiments, the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response when administered to the SCS is less than the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response when administered intravitreously. In some embodiments, the pharmaceutical composition has the same vector genome concentration when administered to the SCS as when administered via subretinal administration or via intravitreous administration. In some embodiments, the pharmaceutical composition has the same amount of genome copies when administered to the SCS as when administered via subretinal administration or via intravitreous administration. In some embodiments, the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response in a subject is lower as compared to the effective amount of a reference pharmaceutical composition sufficient to elicit a therapeutic response in the subject when administered to the SCS. In some embodiments, a reference pharmaceutical composition is a pharmaceutical composition before it is diluted. In some embodiments, a reference pharmaceutical composition has higher ionic strength as compared to the pharmaceutical composition. In some embodiments, the pharmaceutical composition has a higher level of viral vector aggregation (e.g., AAV aggregation) as compared to a reference pharmaceutical composition. In some embodiments, the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response when administered to the SCS is less than the effective amount of a reference pharmaceutical composition sufficient to elicit a therapeutic response when administered subretinally. In some embodiments, the effective amount of the pharmaceutical composition sufficient to elicit a therapeutic response when administered to the SCS is less than the effective amount of a reference pharmaceutical composition sufficient to elicit a therapeutic response when administered intravitreously. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same vector genome concentration. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same amount of genome copies.

[0202] Also provided herein are methods of preparing a pharmaceutical composition. In some embodiments, a method of preparing a pharmaceutical composition includes preparing a composition (e.g., a reference pharmaceutical composition) comprising phosphate-buffered saline, sucrose, and a therapeutically effective amount of a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene. In some embodiments, a method of preparing a pharmaceutical composition includes admixing a solution comprising phosphate-buffered saline and sucrose to a composition comprising AAV. In some embodiments, the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the composition (or reference pharmaceutical composition). In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 135 mM. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 40 mM. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 20 mM. In some embodiments, the pharmaceutical composition has substantially the same tonicity or osmolality as the composition.

[0203] Provided herein are methods of preparing a pharmaceutical composition including admixing a solution comprising phosphate-buffered saline and sucrose to a composition (e.g., a composition having a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene). In some embodiments, the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the composition. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 135 mM. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 40 mM. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 20 mM. In some embodiments, the pharmaceutical composition has substantially the same tonicity or osmolality as the composition.

[0204] Also provided herein are kits for preparing a pharmaceutical composition. In some embodiments, a kit includes (i) a composition comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene; and (ii) a solution comprising phosphate-buffered saline and sucrose. In some embodiments, a kit can include instructions for admixing the composition with the solution. In some embodiments, the instructions includes instructions on admixing the solution with the composition to obtain a pharmaceutical composition. In some embodiments, the composition comprises a phosphate-buffered saline and sucrose. In some embodiments, the composition comprises 4% sucrose. In some embodiments, the solution comprises 10% sucrose. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 135 mM. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 40 mM. In some embodiments, the pharmaceutical composition has an ionic strength of about or of at most about 20 mM. In some embodiments, the pharmaceutical composition has substantially the same tonicity or osmolality as the composition.In some embodiments, any of the methods, or the pharmaceutical composition, or the kits of the present disclosure result in at least some of the aggregated recombinant AAV in the pharmaceutical composition to disaggregate after the pharmaceutical composition is administered to the suprachoroidal space of an eye of a human subject. In some embodiments, aggregation of the recombinant AAV is capable of being reversed upon suprachoroidal administration of the pharmaceutical composition to a human subject. In some embodiments, the aggregated recombinant AAVs turn to monomers or become less aggregated once injected in the SCS of a subject. In some embodiments, the composition comprises potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and optionally a surfactant. In some embodiments, the composition comprises modified Dulbecco's phosphate-buffered saline solution, and optionally a surfactant. In some embodiments, the composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, and a surfactant. In some embodiments, the solution comprises a phosphate-buffered sodium chloride and sucrose. In some embodiments, admixing the solution with the composition dilutes the composition by about or at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold. In some embodiments, admixing the solution with the composition occurs on the same day that the pharmaceutical composition is administered to the suprachoroidal space of an eye of a human subject. In some embodiments, admixing the solution with the composition occurs within 24 hours of the pharmaceutical composition being administered to the suprachoroidal space of an eye of a human subject. In some embodiments, the pharmaceutical composition includes about 1.0×1012 to about 3.0×1012 genome copies of the recombinant AAV. In some embodiments, the recombinant AAV includes components from AAV8 and the pharmaceutical composition has an ionic strength between about 30 mM to about 60 mM. In some embodiments, the recombinant AAV includes components from AAV9 and the pharmaceutical composition has an ionic strength between about 15 mM to about 30 mM. In some embodiments, the recombinant AAV includes components from AAV2 and the pharmaceutical composition has an ionic strength between about 100 mM to about 200 mM.

[0205] In some embodiments, the pharmaceutical composition is substantially localized near the insertion site (see Section 4.2.1 and Section 4.2.2). In some embodiments, the pharmaceutical composition results in a higher level of transgene expression (concentration) when the pharmaceutical composition is administered in the SCS as compared to when the pharmaceutical composition is administered subretinally or intravitreously (see Section 4.2.5). In some embodiments, the pharmaceutical composition results in a higher level of transgene expression (concentration) when the pharmaceutical composition is administered in the SCS as compared to when a reference pharmaceutical composition is administered subretinally, intravitreously, or in the SCS (see Section 4.2.5). In some embodiments, the pharmaceutical composition results in a higher level of AAV when the pharmaceutical composition is administered in the SCS as compared to when the pharmaceutical composition is administered subretinally or intravitreously (see Section 4.2.4). In some embodiments, the pharmaceutical composition results in a higher level of AAV when the pharmaceutical composition is administered in the SCS as compared to when a reference pharmaceutical composition is administered subretinally, intravitreously, or in the SCS (see Section 4.2.4). In some embodiments, the pharmaceutical composition results in a higher rate of transduction (rate of infection) at a site of injection when the pharmaceutical composition is administered in the SCS as compared to when the pharmaceutical composition is administered subretinally or intravitreously (see Section 4.2.4). In some embodiments, the pharmaceutical composition results in a higher rate of transduction (rate of infection) at a site of injection when the pharmaceutical composition is administered in the SCS as compared to when a reference pharmaceutical composition is administered subretinally, intravitreously, or in the SCS (see Section 4.2.4). In some embodiments, the pharmaceutical composition results in reduced vasodilation and / or vascular leakage when the pharmaceutical composition is administered in the SCS as compared to when the pharmaceutical composition is administered subretinally or intravitreously. In some embodiments, the pharmaceutical composition results in reduced vasodilation and / or vascular leakage when the pharmaceutical composition is administered in the SCS as compared to when a reference pharmaceutical composition is administered subretinally, intravitreously, or in the SCS. In some embodiments, the reference pharmaceutical composition includes the recombinant adeno-associated virus (AAV) vector comprising the expression cassette encoding the transgene. In some embodiments, the pharmaceutical composition has a higher level of AAV aggregation than the reference pharmaceutical composition. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same vector genome concentration. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same amount of genome copies.4.1.1 Dilution of Ionic Strength

[0206] In some embodiments, a pharmaceutical composition is diluted with a solution containing a lower ionic strength to reduce the ionic strength of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is diluted prior to administration. In some embodiments, the pharmaceutical composition is diluted and stored. In some embodiments, a solution containing lower ionic strength as compared to the pharmaceutical composition is added to the pharmaceutical composition to provide pharmaceutical compositions comprising 5%, 10%, 15%, 20%, 25%, 30%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% the ionic strength of the undiluted pharmaceutical composition (e.g., diluted two-times, three-times, four-times, eight-times, or ten-times). In some embodiments, the pharmaceutical composition is diluted with a phosphate-buffered 10% sucrose solution. In some embodiments, the pharmaceutical composition comprises modified DPBS with 4% sucrose. In some embodiments, the pharmaceutical composition comprises a poloxamer (e.g., P188).

[0207] In some embodiments, the solutions containing lower ionic strength and that are used to dilute the pharmaceutical composition comprises a lower amount or concentration of a salt as compared to the undiluted pharmaceutical composition. Examples of salts include, but are not limited to, sodium chloride, sodium sulfate, and ammonium sulfate.

[0208] In some embodiments, the solutions containing lower ionic strength and used for dilutions comprise about or at most about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the salt concentration in the undiluted pharmaceutical composition. In some embodiments, the solutions containing lower ionic strength and used for dilutions does not comprise a salt. In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions comprise about or at most about 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, or 600 mM of a salt (e.g., NaCl). In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions does not comprise salt. In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions has a salt concentration of about 0 mM to about 30 mM, 0 mM to about 25 mM, 0 mM to about 100 mM, 0 mM to about 50 mM, 0 mM to about 200 mM, 5 mM to about 100 mM, 10 mM to about 30 mM, 10 mM to about 40 mM, 10 mM to about 50 mM, 10 mM to about 60 mM, 10 mM to about 100 mM, 5 mM to about 50 mM, 5 mM to about 30 mM, 1 mM to about 100 mM, 1 mM to about 40 mM, or 1 mM to about 30 mM, 1 mM to about 200 mM, 1 mM to about 600 mM, or 1 mM to about 300 mM. In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions comprise about or at most about 10 mM of salt. In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions comprise about or at most about 100 mM of salt. In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions comprise about or at most about 200 mM of salt. In some embodiments, the solutions containing lower ionic strength and used for dilutions comprises sucrose. In some embodiments, the solutions containing lower ionic strength and used for dilutions comprises 4%, 6%, 8%, 10%, 15%, 20%, 25%, 30% (or higher) sucrose. In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions comprises 4% sucrose. In some embodiments, the solution used for dilutions comprises 6% sucrose. In some embodiments, the solution used for dilutions comprises 10% sucrose. In some embodiments, the pharmaceutical composition comprises about, at least about, or at most about: 0.5% (w / v), 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, or higher than 40% sucrose.

[0209] In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions has an ionic strength of about or at most about 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, or 600 mM. In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions has an ionic strength of about or at most about 25 mM. In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions has an ionic strength of about or at most about 50 mM. In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions has an ionic strength of about or at most about 15 mM. In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions has an ionic strength of about or at most about 80 mM. In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions has an ionic strength of about or at most about 100 mM. In some embodiments, the solution (e.g., solutions containing lower ionic strength) used for dilutions has an ionic strength of about 5 mM to about 100 mM, 10 mM to about 30 mM, 10 mM to about 40 mM, 10 mM to about 50 mM, 10 mM to about 60 mM, 10 mM to about 100 mM, 5 mM to about 50 mM, 5 mM to about 30 mM, 1 mM to about 100 mM, 1 mM to about 40 mM, or 1 mM to about 30 mM, 1 mM to about 200 mM, 1 mM to about 600 mM, or 1 mM to about 300 mM.

[0210] In some embodiments, a low ionic strength pharmaceutical composition is used to administer an AAV encoding a transgene. In some embodiments, a pharmaceutical composition (e.g., diluted liquid formulation) having a reduced ionic strength as compared to a reference is used to administer an AAV encoding a transgene. In some embodiments, a low salt pharmaceutical composition (e.g., diluted liquid formulation) is used to administer an AAV encoding a transgene. In some embodiments, a pharmaceutical composition having a lower salt concentration as compared to a control solution, or as compared to a reference pharmaceutical composition, or as compared to PBS, or as compared to a commonly used pharmaceutical composition for subretinal injection, is used to administer an AAV encoding a transgene.

[0211] In some embodiments, examples of compounds that can be used to prepare a salt includes (but not limited to) aluminum, acetate, glutamate, mucate, arginine, aspartate, glycolate, napsylate, benzathine, benzenesulfonate, glycollylarsanilate, nitrate, calcium, benzoate, hexanoate, octanoate, chloroprocaine, besylate, hexylresorcinate, oleate, choline, bicarbonate, hydrabamine, pamoate, diethanolamine, bitartrate, hydroxynaphthoate, pantothenate, ethanolamine, bromide, iodide, phosphate, ethylenediamine, camsylate, isethionate, polygalacturonate, histidine, carbonate, isethionate, propionate, lithium, chloride, lactate, salicylate, lysine, citrate, lactobionate, stearate, magnesium, decanoate, malate, subacetate, meglumine, edetate, maleate, succinate, potassium, estolate, mandelate, sulfate, procaine, esylate, mesylate, tartrate, sodium, fumarate, methylbromide, teoclate, trimethylamine, gluceptate, methylnitrate, tosylate, zinc, gluconate, methylsulfate, and triethiodide.

[0212] In some embodiments, a salt in a solution or to be used in a pharmaceutical composition includes, but it is not limited to, sodium fluoride, sodium chloride, sodium bromide, sodium iodide, sodium sulfate, sodium bicarbonate, sodium carbonate, sodium amide (NaNH2), or any salt suitable or pharmaceutical formulation.4.1.2 Other Components of the Formulation

[0213] In some embodiments, the disclosure provides a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) comprising a recombinant adeno-associated virus (AAV) and at least one of potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and surfactant. In some embodiments, the pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) does not comprise sucrose.

[0214] In some embodiments, the disclosure provides a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) and at least one of an ionic salt excipient or buffering agent, sucrose, and surfactant. In some embodiments, the ionic salt excipient or buffering agent can be one or more components from the group consisting of potassium phosphate monobasic, potassium phosphate, sodium chloride, sodium phosphate dibasic anhydrous, sodium phosphate hexahydrate, sodium phosphate monobasic monohydrate, tromethamine, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), amino acid, histidine, histidine hydrochloride (histidine-HCl), sodium succinate, sodium citrate, sodium acetate, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), sodium sulfate, magnesium sulfate, magnesium chloride 6-hydrate, calcium sulfate, potassium chloride, calcium chloride, and calcium citrate. In some embodiments, the surfactant can be one or more components from the group consisting of poloxamer 188, polysorbate 20, and polysorbate 80.

[0215] In some embodiments, the pharmaceutical composition comprises about, at least about, or at most about: 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1 mg / mL, 1.05 mg / mL, 1.10 mg / mL, 1.15 mg / mL, 1.20 mg / mL, 1.25 mg / mL, 1.30 mg / mL, 1.35 mg / mL, 1.40 mg / mL, 1.45 mg / mL, 1.50 mg / mL, or more than 1.50 mg / mL of sodium phosphate dibasic anhydrous (or an equivalent). In some embodiments, the pharmaceutical composition comprises about, at least about, or at most about: 4.5 mg / mL, 4.55 mg / mL, 4.6 mg / mL, 4.65 mg / mL, 4.7 mg / mL, 4.75 mg / mL, 4.8 mg / mL, 4.85 mg / mL, 4.9 mg / mL, 4.95 mg / mL, 5 mg / mL, 5.05 mg / mL, 5.10 mg / mL, 5.15 mg / mL, 5.20 mg / mL, 5.25 mg / mL, 5.30 mg / mL, 5.35 mg / mL, 5.40 mg / mL, 5.45 mg / mL, 5.50 mg / mL, 5.55 mg / mL, 5.60 mg / mL, 5.65 mg / mL, 5.70 mg / mL, 5.75 mg / mL, 5.80 mg / mL, 5.81 mg / mL, 5.82 mg / mL, 5.83 mg / mL, 5.84 mg / mL, 5.85 mg / mL, 5.86 mg / mL, 5.87 mg / mL, 5.88 mg / mL, 5.89 mg / mL, 5.90 mg / mL, 5.95 mg / mL, 6 mg / mL, or more than 6 mg / mL of sodium chloride (or an equivalent). In some embodiments, the pharmaceutical composition comprises about, at least about, or at most about: 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, or more than 1 mg / mL of potassium chloride and / or potassium phosphate monobasic (or equivalents thereof).

[0216] In certain embodiments, the pharmaceutical composition has an ionic strength of about 60 mM to about 115 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 60 mM to about 100 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 65 mM to about 95 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 70 mM to about 90 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 75 mM to about 85 mM.

[0217] In certain embodiments, the pharmaceutical composition has an ionic strength of about 30 mM to about 100 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 35 mM to about 95 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 40 mM to about 90 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 45 mM to about 85 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 50 mM to about 80 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 55 mM to about 75 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 60 mM to about 70 mM.

[0218] In certain embodiments, the pharmaceutical composition comprises potassium chloride (e.g., at a concentration of 0.2 g / L). In certain embodiments, the pharmaceutical composition comprises potassium phosphate monobasic (e.g., at a concentration of 0.2 g / L). In certain embodiments, the pharmaceutical composition comprises sodium chloride (e.g., at a concentration of 5.84 g / L). In certain embodiments, the pharmaceutical composition comprises sodium phosphate dibasic anhydrous (e.g., at a concentration of 1.15 g / L). In certain embodiments, the pharmaceutical composition comprises potassium chloride, potassium phosphate monobasic, sodium chloride, and sodium phosphate dibasic anhydrous.

[0219] In some embodiments, the reference pharmaceutical composition comprises the same components as the pharmaceutical composition. In some embodiments, the reference pharmaceutical composition comprises the same components as the pharmaceutical composition but has lower ionic strength than the pharmaceutical composition. In some embodiments, the reference pharmaceutical composition comprises the same components as the pharmaceutical composition with the exception of one or more components that affect or increase ionic strength of a composition or solution.

[0220] In certain embodiments, the pharmaceutical composition comprises sucrose at a concentration of 10% (weight / volume, 30 g / L) to 18% (weight / volume, 180 g / L). In certain embodiments, the pharmaceutical composition comprises sucrose at a concentration of 4% (weight / volume, 40 g / L).

[0221] In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.001% (weight / volume, 0.01 g / L). In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0005% (weight / volume, 0.005 g / L) to 0.05% (weight / volume, 0.5 g / L). In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.001% (weight / volume, 0.01 g / L). In certain embodiments, the pharmaceutical composition comprises polysorbate 20 at a concentration of 0.0005% (weight / volume, 0.05 g / L) to 0.05% (weight / volume, 0.5 g / L). In certain embodiments, the pharmaceutical composition comprises polysorbate 80 at a concentration of 0.0005% (weight / volume, 0.05 g / L) to 0.05% (weight / volume, 0.5 g / L). In some embodiments, the pharmaceutical composition comprises a surfactant (e.g., poloxamer 188, polysorbate 20, and / or polysorbate 80) at a concentration of about, at least about, or at most about: 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 0.1%, or more than 0.1%.

[0222] In certain embodiments, the pH of the pharmaceutical composition is about 7.4. In certain embodiments, the pH of the pharmaceutical composition is about 6.0 to 9.0. In certain embodiments, the pH of the pharmaceutical composition is 7.4. In certain embodiments, the pH of the pharmaceutical composition is 6.0 to 9.0.

[0223] In certain embodiments, the pharmaceutical composition is in a hydrophobically-coated glass vial. In certain embodiments, the pharmaceutical composition is in a Cyclo Olefin Polymer (COP) vial. In certain embodiments, the pharmaceutical composition is in a Daikyo Crystal Zenith® (CZ) vial. In certain embodiments, the pharmaceutical composition is in a TopLyo coated vial.

[0224] In certain embodiments, disclosed herein is a pharmaceutical composition comprising a recombinant AAV and at least one of: (a) potassium chloride at a concentration of 0.2 g / L, (b) potassium phosphate monobasic at a concentration of 0.2 g / L, (c) sodium chloride at a concentration of 5.84 g / L, (d) sodium phosphate dibasic anhydrous at a concentration of 1.15 g / L, (e) sucrose at a concentration of 4% weight / volume (40 g / L), (f) poloxamer 188 at a concentration of 0.001% weight / volume (0.01 g / L), and (g) water, and wherein the recombinant AAV is AAV8. In some embodiments, the pharmaceutical composition does not comprise sucrose. In certain embodiments, disclosed herein is a pharmaceutical composition comprising a composition comprising recombinant AAV and a solution comprising 10% Sucrose, 2.70 mM potassium chloride, 8.10 mM sodium phosphate dibasic anhydrous, 1.47 mM potassium phosphate monobasic, 292 mM sucrose, 0.001% (0.01 mg / mL) poloxamer 188, pH 7.4, wherein, for example, the composition and solution are admixed at a composition to solution ratio of 1 to 9. In some embodiments, disclosed herein is a method for preparing a pharmaceutical composition by admixing the composition and solution at a composition to solution ratio of 1 to 9. In some embodiments, disclosed herein is a kit used for preparing a pharmaceutical composition by admixing the composition and solution at a composition to solution ratio of 1 to 9. In some embodiments, the composition and solution are admixed at a composition to solution ratio of 1 to 1, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 12, 1 to 15, 1 to 17, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, or more than 1 to 40.

[0225] In some embodiments, the pharmaceutical composition comprises (a) an AAV8 or AAV9 that encodes Tripeptidyl-Peptidase 1 and at least one of: (b) potassium chloride at a concentration of 0.2 g / L, (c) potassium phosphate monobasic at a concentration of 0.2 g / L, (d) sodium chloride at a concentration of 5.84 g / L, (e) sodium phosphate dibasic anhydrous at a concentration of 1.15 g / L, (f) sucrose at a concentration of 4% weight / volume (40 g / L), (g) poloxamer 188 at a concentration of 0.001% weight / volume (0.01 g / L), and (h) water. In some embodiments, the pharmaceutical composition does not comprise sucrose. In some embodiments, the level of AAV aggregation in the pharmaceutical composition impacts Batten-CLN2-associated vision loss.

[0226] In some embodiments, the pharmaceutical composition has desired viscosity, density, and / or osmolality that is suitable for suprachoroidal injection (for example, via a suprachoroidal drug delivery device such as a microinjector with a microneedle). In some embodiments, the pharmaceutical composition is a liquid composition. In some embodiments, the pharmaceutical composition is a frozen composition. In some embodiments, the pharmaceutical composition is a lyophilized composition from a liquid composition disclosed herein. In some embodiments, the pharmaceutical composition is a reconstituted lyophilized formulation.

[0227] In some embodiments, the pharmaceutical composition is a lyophilized composition comprising a residual moisture content between about 1% and about 7%. In some embodiments, the pharmaceutical composition is a lyophilized composition comprising a residual moisture content between about 2% and about 6%. In some embodiments, the pharmaceutical composition is a lyophilized composition comprising a residual moisture content between about 10% and about 4%. In some embodiments, the pharmaceutical composition is a lyophilized composition comprising a residual moisture content of about 5%.

[0228] In certain embodiments, the pharmaceutical composition has a osmolality range of 200 mOsm / L to 660 mOsm / L. In certain embodiments, the pharmaceutical composition has a osmolality of about, of at least about, or of at most about: 200 mOsm / L, 250 mOsm / L, 300 mOsm / L, 350 mOsm / L, 400 mOsm / L, 450 mOsm / L, 500 mOsm / L, 550 mOsm / L, 600 mOsm / L, 650 mOsm / L, or 660 mOsm / L.

[0229] In certain embodiments, gene therapy constructs are supplied as a frozen sterile, single use solution of the AAV vector active ingredient in a formulation buffer. In a specific embodiment, the pharmaceutical compositions suitable for suprachoroidal administration comprise a suspension of the recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients. In some embodiments, the construct is formulated in Dulbecco's phosphate buffered saline and 0.001% poloxamer 188, pH=7.4.4.2 Functional Properties4.2.1 Clearance Time

[0230] The disclosure provides a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) resulting in a delayed clearance time from the SCS. In some embodiments, a pharmaceutical composition comprising aggregated AAV or comprising more levels of aggregated AAV results in delayed clearance time from the SCS as compared to a pharmaceutical composition comprising lower levels of aggregated AAV, or comprising substantially no aggregated AAV, or comprising no aggregation. In some embodiments, a pharmaceutical composition comprising aggregated AAV or comprising more levels of aggregated AAV results in delayed clearance time from the eye as compared to a pharmaceutical composition comprising lower levels of aggregated AAV, or comprising substantially no aggregated AAV, or comprising no aggregation. In some embodiments, a pharmaceutical composition comprises more aggregated AAV than a reference composition normally used for subretinal injection. In some embodiments, the clearance time of the pharmaceutical composition after the pharmaceutical composition is administered to the SCS is equal to or higher than the clearance time of a reference pharmaceutical composition after the reference pharmaceutical composition is administered subretinally or intravitreously. In some embodiments, the clearance time of the pharmaceutical composition after the pharmaceutical composition is administered to the SCS is equal to or higher than the clearance time of a reference pharmaceutical composition after the reference pharmaceutical composition is administered to the SCS. In some embodiments, the pharmaceutical composition has more aggregated AAV than the level of aggregated AAV in the reference pharmaceutical composition.

[0231] In some embodiments, a pharmaceutical composition (e.g., diluted formulation or low ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in a clearance time from the SCS of about 30 minutes to about 20 hours, about 2 hours to about 20 hours, about 30 minutes to about 24 hours, about 1 hour to about 2 hours, about 30 minutes to about 90 days, about 30 minutes to about 60 days, about 30 minutes to about 30 days, about 30 minutes to about 21 days, about 30 minutes to about 14 days, about 30 minutes to about 7 days, about 30 minutes to about 3 days, about 30 minutes to about 2 days, about 30 minutes to about 1 day, about 4 hours to about 90 days, about 4 hours to about 60 days, about 4 hours to about 30 days, about 4 hours to about 21 days, about 4 hours to about 14 days, about 4 hours to about 7 days, about 4 hours to about 3 days, about 4 hours to about 2 days, about 4 hours to about 1 day, about 4 hours to about 8 hours, about 4 hours to about 16 hours, about 4 hours to about 20 hours, about 1 day to about 90 days, about 1 day to about 60 days, about 1 day to about 30 days, about 1 day to about 21 days, about 1 day to about 14 days, about 1 day to about 7 days, about 1 day to about 3 days, about 2 days to about 90 days, about 3 days to about 90 days, about 3 days to about 60 days, about 3 days to about 30 days, about 3 days to about 21 days, about 3 days to about 14 days, or about 3 days to about 7 days. In some embodiments, the clearance time from the SCS is of about 3 days to about 365 days, about 3 days to about 300 days, about 3 days to about 200 days, about 3 days to about 150 days, about 3 days to about 125 days, about 7 days to about 365 days, about 7 days to about 300 days, about 7 days to about 200 days, about 7 days to about 150 days, about 7 days to about 125 days. The “clearance time from the SCS” is the time required for substantially all of the pharmaceutical composition, the pharmaceutical agent, or the AAV to escape the SCS. In some embodiments, the “clearance time from the SCS” is the time required for the pharmaceutical composition, the pharmaceutical agent, or the AAV to not be detected in the SCS by any standard method (such as those described in Section 4.6 and Section 5). In some embodiments, the “clearance time from the SCS” is when the pharmaceutical composition, the pharmaceutical agent, or the AAV is present in the SCS in an amount that is at most about 2% or at most about 5% as detected by any standard method (such as those described in Section 4.6 and Section 5).

[0232] In some embodiments, the pharmaceutical composition (e.g., diluted formulation or low ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in a clearance time from the eye of about 30 minutes to about 20 hours, about 2 hours to about 20 hours, about 30 minutes to about 24 hours, about 1 hour to about 2 hours, about 30 minutes to about 90 days, about 30 minutes to about 60 days, about 30 minutes to about 30 days, about 30 minutes to about 21 days, about 30 minutes to about 14 days, about 30 minutes to about 7 days, about 30 minutes to about 3 days, about 30 minutes to about 2 days, about 30 minutes to about 1 day, about 4 hours to about 90 days, about 4 hours to about 60 days, about 4 hours to about 30 days, about 4 hours to about 21 days, about 4 hours to about 14 days, about 4 hours to about 7 days, about 4 hours to about 3 days, about 4 hours to about 2 days, about 4 hours to about 1 day, about 4 hours to about 8 hours, about 4 hours to about 16 hours, about 4 hours to about 20 hours, about 1 day to about 90 days, about 1 day to about 60 days, about 1 day to about 30 days, about 1 day to about 21 days, about 1 day to about 14 days, about 1 day to about 7 days, about 1 day to about 3 days, about 2 days to about 90 days, about 3 days to about 90 days, about 3 days to about 60 days, about 3 days to about 30 days, about 3 days to about 21 days, about 3 days to about 14 days, or about 3 days to about 7 days. In some embodiments, the clearance time from the eye is of about 3 days to about 365 days, about 3 days to about 300 days, about 3 days to about 200 days, about 3 days to about 150 days, about 3 days to about 125 days, about 7 days to about 365 days, about 7 days to about 300 days, about 7 days to about 200 days, about 7 days to about 150 days, about 7 days to about 125 days. The “clearance time from the eye” is the time required for substantially all of the pharmaceutical composition, the pharmaceutical agent, or the AAV to escape the eye. In some embodiments, the “clearance time from the eye” is the time required for the pharmaceutical composition, the pharmaceutical agent, or the AAV to not be detected in the eye by any method (such as those described in Section 4.6 and Section 5). In some embodiments, the “clearance time from the eye” is when the pharmaceutical composition, the pharmaceutical agent, or the AAV is present in the eye in an amount that is at most about 2% or at most about 5% as detected by any standard method (such as those described in Section 4.6 and Section 5).

[0233] In some embodiments, the clearance time is not prior to (e.g., the clearance time from the SCS or the eye does not occur before) about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days after administration of the pharmaceutical composition (e.g., a liquid formulation). In some embodiments, the clearance time is about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days after administration of the pharmaceutical composition (e.g., diluted formulation or low ionic strength formulation).

[0234] In some embodiments, a pharmaceutical composition comprising AAV aggregation or a higher level of AAV aggregation (e.g., diluted formulation or low ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in a clearance time that is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater than when a pharmaceutical composition (reference pharmaceutical composition) that does not comprise AAV aggregation or comprises a lower level of AAV aggregation is used to administer the AAV comprising the expression cassette encoding the transgene (e.g., via a subretinal administration, intravitreous administration, or to the SCS).

[0235] In some embodiments, a suprachoroidal administration of a pharmaceutical composition comprising AAV aggregation or a higher level of AAV aggregation (e.g., diluted formulation or low ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in a clearance time that is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater than when a pharmaceutical composition (reference pharmaceutical composition) that does not comprise AAV aggregation or comprises a lower level of AAV aggregation is used, for example, to administer the AAV comprising the expression cassette encoding the transgene by suprachoroidal administration.

[0236] In some embodiments, a suprachoroidal administration of a pharmaceutical composition comprising AAV aggregation or a higher level of AAV aggregation (e.g., diluted formulation or low ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in a clearance time that is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater than when a pharmaceutical composition (reference pharmaceutical composition) that does not comprise AAV aggregation or comprises a lower level of AAV aggregation is used, for example, to administer the AAV comprising the expression cassette encoding the transgene by subretinal administration or by intravitreous administration.

[0237] In some embodiments, a suprachoroidal administration a pharmaceutical composition comprising AAV aggregation or a higher level of AAV aggregation as compared to a reference (e.g., diluted formulation or low ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in a clearance time that is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater than when the same pharmaceutical composition is used, for example, to administer the AAV comprising the expression cassette encoding the transgene via subretinal administration or via intravitreous administration.

[0238] In some embodiments, the clearance time of a pharmaceutical composition comprising aggregated AAV (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) administered by suprachoroidal injection is greater than the clearance time of the same pharmaceutical composition administered via subretinal administration or via intravitreous administration. In some embodiments, the clearance time after a pharmaceutical composition comprising aggregated AAV (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) is administered by suprachoroidal injection is greater than the clearance time after a comparable pharmaceutical composition comprising no AAV aggregation or lower levels of AAV aggregation (e.g., a reference pharmaceutical composition) is administered by suprachoroidal injection. In some embodiments, the clearance time of a pharmaceutical composition comprising aggregated AAV (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) after suprachoroidal injection is greater than the clearance time of a comparable pharmaceutical composition comprising lower levels of AAV aggregation (or no detectable AAV aggregation) after subretinal administration or via intravitreous administration. In some embodiments, the clearance time of a pharmaceutical composition comprising aggregated AAV (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) administered by suprachoroidal injection is greater than a comparable pharmaceutical composition comprising lower levels of AAV aggregation (or no detectable AAV aggregation) administered via subretinal administration or via intravitreous administration.

[0239] In some embodiments, the clearance time of a pharmaceutical composition comprising aggregated AAV (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) after suprachoroidal administration is greater than the clearance time of the same pharmaceutical composition after subretinal administration or intravitreous administration by at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days.

[0240] In some embodiments, the clearance time of a pharmaceutical composition comprising aggregated AAV (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) after suprachoroidal administration is greater than the clearance time after a comparable pharmaceutical composition comprising less aggregated AAV or no detectable aggregated AAV is administered by suprachoroidal injection by at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days.

[0241] In some embodiments, the clearance time of a pharmaceutical composition comprising aggregated AAV (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) after suprachoroidal administration is greater than the clearance time after a comparable pharmaceutical composition comprising less aggregated AAV or no detectable aggregated AAV is administered by subretinal administration or intravitreous administration by at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days.

[0242] In some embodiments, the clearance time of the pharmaceutical composition administered via intravitreous injection or via subretinal injection is of at most about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or at most about 400 days after administration.

[0243] In some embodiments, the clearance time of a reference pharmaceutical composition administered by intravitreous injection, subretinal injection, or to the SCS is of at most about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or at most about 400 days after administration.

[0244] In some embodiments, the clearance time is the clearance time from the eye. In some embodiments, the clearance time is the clearance time from the SCS. In some embodiments, the clearance time is the clearance time from the site of injection.

[0245] In some embodiments, the “clearance time from the SCS” is the amount of time required following injection of the pharmaceutical composition, the pharmaceutical agent, or the AAV required for the SCS thickness at or near the site of injection to decrease to about 1 nm or less, about 2 nm or less, about 5 nm or less, about 10 nm or less, about 25 nm or less, about 50 nm or less, about 100 nm or less, about 200 nm or less, or about 500 nm or less, as measured by standard techniques (e.g. in-vivo imaging techniques such as OCT imaging, ultra-high resolution OCT (UHR-OCT), ultrasound and three-dimensional (3D) cryo-reconstruction). In some embodiments, the “clearance time from the SCS” is the amount of time required following injection of the pharmaceutical composition, the pharmaceutical agent, or the AAV required for the SCS thickness at or near the site of injection to decrease to 500 nm or less, about 200 nm or less, about 100 nm or less, about 50 nm or less, about 25 nm or less, about 10 nm or less, or is undetectable, as measured by standard techniques (e.g. in-vivo imaging techniques such as OCT imaging, UHR-OCT, ultrasound and three-dimensional (3D) cryo-reconstruction). In some embodiments, SCS thickness is measured using Heidelberg Optical Coherence Tomography (OCT). In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation sufficient to make the clearance time at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days.

[0246] In some embodiments, the “clearance time from the SCS” is the amount of time required following injection for the pharmaceutical composition, the pharmaceutical agent, or the AAV required to spread circumferentially from the site of injection to cover about one-sixteenth or more, about one-eighth or more, about one-fourth or more, about one-half or more, about three-fourths or more, or all of the circumference of the choroid, as measured by standard techniques (e.g. in-vivo imaging techniques such as OCT imaging). In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation sufficient to make the clearance time at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days.

[0247] In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachroidal space of the eye of a pig (e.g., minipig, such as Yucatan minipig). In some embodiments, the pig is a minipig. In some embodiments, the minipig can be Goettingen, Yucatan, Bama Xiang Zhu, Wuzhishan, and / or Xi Shuang Banna. In some embodiments, the minipig is Yucatan. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation such that, when administered to the SCS of the eye of a pig, the clearance time of the pharmaceutical composition is between about 5 days and about 15 days, about 6 days to about 15 days days, about 7 days to about 15 days, about 8 days to about 15 days, about 9 days to about 15 days, about 10 days to about 15 days, about 11 days to about 15 days, about 12 days to about 15 days, about 13 days to about 15 days, about 14 days to about 15 days, about 5 days to about 14 days, about 5 days to about 13 days, about 5 days to about 12 days, about 5 days to about 11 days, about 5 days to about 10 days, about 5 days to about 9 days, about 5 days to about 8 days, about 5 days to about 7 days, or about 5 days to about 6 days. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachroidal space of the eye of a pig. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation such that, when administered to the SCS of the eye of a pig, the clearance time of the pharmaceutical composition is between about 5 days and about 15 days.4.2.2 Circumferential Spread

[0248] In some embodiments, a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) localizes at the site of injection. In some embodiments, a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) localizes at the site of injection for a longer period of time than a reference pharmaceutical composition comprising less AAV aggregation or no detectable AAV aggregation. In some embodiments, a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) localizes at the site of injection for a longer period of time when injected in the SCS as compared to when the pharmaceutical composition is administered by subretinal injection or intravitreous injection. The pharmaceutical composition can have different levels of viral vector aggregation. In some embodiments, a pharmaceutical composition comprising aggregated AAV remains localized in the SCS for a longer period of time as compared to a pharmaceutical composition comprising less AAV aggregation or no detectable AAV aggregation (e.g., a reference pharmaceutical composition).

[0249] In some embodiments, localization can be determined by evaluating circumferential spread (e.g., 2D circumferential spread). In some embodiments, circumferential spread is determined by analyzing SCS expansion or opening in the quadrant where the injection was made (in some cases, in 2D this space adjacent to the choroid appears linear). In some embodiments, a pharmaceutical composition comprising aggregated AAV (e.g., diluted formulation or lower ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in a circumferential spread that is at least 2 times less, at least 3 times less, at least 4 times less, at least 5 times less, at least 6 times less, at least 7 times less, at least 8 times less, at least 9 times less, at least 10 times less, at least 15 times less, at least 20 times less, at least 50 times less, at least 100 times less, at least 5% less, at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40%, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, at least 100% less, at least 150% less, or at least 200% less, at least 250% less, or at least 300%, at least 400% less, or at least 500% less than when a reference pharmaceutical composition (e.g., comprising less AAV aggregation or no detectable levels of AAV aggregation) is used to administer the AAV comprising the expression cassette encoding the transgene (e.g., by suprachoroidal injection, by subretinal injection, or by intravitreous injection).

[0250] In some embodiments, a suprachoroidal administration of a pharmaceutical composition comprising aggregated AAV (e.g., diluted formulation or lower ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in a circumferential spread that is at least 2 times less, at least 3 times less, at least 4 times less, at least 5 times less, at least 6 times less, at least 7 times less, at least 8 times less, at least 9 times less, at least 10 times less, at least 15 times less, at least 20 times less, at least 50 times less, at least 100 times less, at least 5% less, at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40%, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, at least 100% less, at least 150% less, or at least 200% less, at least 250% less, or at least 300%, at least 400% less, or at least 500% less than when reference pharmaceutical composition (e.g., comprising less AAV aggregation or no detectable levels of AAV aggregation) is used, for example, to administer the AAV comprising the expression cassette encoding the transgene by suprachoroidal administration, by subretinal administration, or by intravitreous administration.

[0251] In some embodiments, a suprachoroidal administration of a pharmaceutical composition comprising aggregated AAV (e.g., diluted formulation or lower ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in a circumferential spread that is at least 2 times less, at least 3 times less, at least 4 times less, at least 5 times less, at least 6 times less, at least 7 times less, at least 8 times less, at least 9 times less, at least 10 times less, at least 15 times less, at least 20 times less, at least 50 times less, at least 100 times less, at least 5% less, at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40%, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, at least 100% less, at least 150% less, or at least 200% less, at least 250% less, or at least 300%, at least 400% less, or at least 500% less than when the same pharmaceutical composition is used, for example, to administer the AAV comprising the expression cassette encoding the transgene by subretinal administration or by intravitreous administration.

[0252] In some embodiments, the circumferential spread can be determined 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days after the pharmaceutical composition or the reference pharmaceutical composition is administered.

[0253] In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachroidal space of the eye of a pig. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation such that, when administered to the SCS of the eye of a pig, the circumferential spread of the pharmaceutical composition from the site of injection is about one-sixteenth or less, about one-eighth or less, about one-fourth or less, or about one-half or less of a surface of the choroid at a time within about 5 minutes, about 10 minutes, 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about one hour, about 2 hours, about 4 hours, about 8 hours, or about 24 hours of administration. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachroidal space of the eye of a pig. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation such that, when administered to the SCS of the eye of a pig, the circumferential spread of the pharmaceutical composition from the site of injection is about about one-eighth or less of a surface of the choroid at a time within about 5 minutes, about 10 minutes, 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about one hour, about 2 hours, about 4 hours, about 8 hours, or about 24 hours of administration. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation such that, when administered to the SCS of the eye of a pig, the circumferential spread of the pharmaceutical composition from the site of injection is about one-eighth or less of a surface of the choroid at a time within about one hour of administration.4.2.3 SCS Thickness

[0254] In some embodiments, localization can be determined by evaluating SCS thickness after a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) is administered to a subject. In some embodiments, a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) increases the thickness of the SCS after the pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) is injected in the SCS. In some embodiments, the infusion into the SCS of a pharmaceutical composition comprising aggregated AAV (e.g., diluted formulation or lower ionic strength formulation) can expand SCS thickness beyond the SCS thickness achieved when a reference pharmaceutical composition (e.g., comprising lower levels of AAV aggregation or comprising no detectable AAV aggregation) is infused into the SCS. In some embodiments, increasing the SCS thickness with a pharmaceutical composition comprising aggregated AAV (e.g., diluted formulation or lower ionic strength formulation) may ease access to the SCS, thereby easing or permitting the disposal of a device in the SCS. In some embodiments, expanding the SCS thickness allows for the pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) and / or the AAV encoded transgene to remain at the site of injection (localized) for a longer period of time. In some embodiments, a pharmaceutical composition comprising aggregated AAV increases the thickness at or near the site of injection for a longer period of time as compared to a reference pharmaceutical composition. In some embodiments, a pharmaceutical composition comprising aggregated AAV increases the thickness at or near the site of injection for a longer period of time as compared to a pharmaceutical composition comprising less AAV aggregation or no detectable level of AAV aggregation. In some embodiments, the thickness at the site of injection after the pharmaceutical composition is administered to the SCS is equal to or higher than the thickness at the site of injection of a reference pharmaceutical composition after the reference pharmaceutical composition is administered subretinally or intravitreously. In some embodiments, the thickness at the site of injection of the pharmaceutical composition after the pharmaceutical composition is administered to the SCS is equal to or higher than the thickness at the site of injection of a reference pharmaceutical composition after the reference pharmaceutical composition is administered to the SCS.

[0255] In some embodiments, a suprachoroidal administration of a pharmaceutical composition comprising aggregated AAV (e.g., diluted formulation or lower ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in an increase in the SCS thickness that is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater than when a reference pharmaceutical composition (comprising lower levels of AAV aggregation or no detectable AAV aggregation) is used, for example, to administer the AAV comprising the expression cassette encoding the transgene by suprachoroidal administration.

[0256] In some embodiments, a suprachoroidal administration of a pharmaceutical composition comprising AAV aggregation (e.g., diluted formulation or lower ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in an increase in thickness at or near the site of injection that is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater than when a reference pharmaceutical composition (comprising lower levels of AAV aggregation or no detectable AAV aggregation) is used, for example, to administer the AAV comprising the expression cassette encoding the transgene by subretinal administration or by intravitreous administration.

[0257] In some embodiments, a suprachoroidal administration of a pharmaceutical composition comprising AAV aggregation (e.g., diluted formulation or lower ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in an increase in thickness at or near the site of injection that is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater than when the same pharmaceutical composition is used, for example, to administer the AAV comprising the expression cassette encoding the transgene by subretinal administration or by intravitreous administration.

[0258] In some embodiments, the thickness obtained at the site of injection after a pharmaceutical composition comprising aggregated AAV (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) is administered by suprachoroidal injection is greater than after a reference pharmaceutical composition is administered by suprachoroidal injection. In some embodiments, the thickness obtained at the site of injection after a pharmaceutical composition comprising aggregated AAV (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) is administered by suprachoroidal injection is greater than after a reference pharmaceutical composition is administered by subretinal injection or by intravitreous injection. In some embodiments, the thickness obtained at the site of injection after a pharmaceutical composition comprising aggregated AAV (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) is administered by suprachoroidal injection is greater than after the same pharmaceutical composition is administered by subretinal administration or by intravitreous administration.

[0259] In some embodiments, the thickness at or near the site of injection (e.g., thickness at or near the SCS) can be determined 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days after the pharmaceutical composition or the reference pharmaceutical composition is administered.

[0260] In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachroidal space of the eye of a pig. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation such that, when administered to the SCS of the eye of a pig, the thickness of the SCS at the site of injection is between about 400 and about 800 μm, about 400 μm to about 700 μm, about 400 μm to about 600 μm, about 400 μm to about 500 μm, about 500 μm to about 800 μm, about 600 μm to about 800 μm, 700 μm to about 800 μm at a time at a time within about 5 minutes, about 10 minutes, 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about one hour, about 2 hours, about 4 hours, about 8 hours, or about 24 hours of administration. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) is administered to the suprachroidal space of the eye of a pig. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation such that, when administered to the SCS of the eye of a pig, the thickness of the SCS at the site of injection is between about 400 and about 800 μm at a time within about 5 minutes, about 10 minutes, 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about one hour, about 2 hours, about 4 hours, about 8 hours, or about 24 hours of administration. In some embodiments, the pharmaceutical composition (e.g., diluted pharmaceutical composition) has an amount of viral vector aggregation such that, when administered to the SCS of the eye of a pig, the thickness of the SCS at the site of injection is between about 400 and about 800 μm at a time within about one hour of administration.4.2.4 Rate of Transduction (Rate of Infection) at Site of Injection

[0261] In some embodiments, the rate of transduction (rate of infection) at the site of injection after a pharmaceutical composition is administered in the SCS is equal to or higher as compared to the rate of transduction (rate of infection) at a site of injection after the same pharmaceutical composition is administered via a subretinal administration or via an intravenous administration. In some embodiments, the rate of transduction (rate of infection) at the site of injection after a pharmaceutical composition is administered in the SCS is equal to or higher as compared to the rate of transduction (rate of infection) at the site of injection after a reference pharmaceutical composition is administered via a subretinal, or intravenous administration, or to the SCS. In some embodiments, the pharmaceutical composition has higher levels of AAV aggregation than the reference pharmaceutical composition. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same vector genome concentration. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same amount of genome copies. In some embodiments, the rate of transduction (rate of infection) at the site of injection after the pharmaceutical composition is administered to the SCS is increased by at least about 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500% as compared to the rate of transduction (rate of infection) at the site of injection after the same pharmaceutical composition is administered via subretinal or via intravitreous administrations. In some embodiments, the rate of transduction (rate of infection) at the site of injection after the pharmaceutical composition is administered to the SCS is increased by at least about 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500% as compared to the rate of transduction (rate of infection) at the site of injection after a reference pharmaceutical composition is administered to the SCS, or via subretinal, or via intravitreous administrations. In some embodiments, the pharmaceutical composition has a higher level of AAV aggregation as compared to the reference pharmaceutical composition.

[0262] In some embodiments, a level of AAV at the site of injection is equal to or higher after the pharmaceutical composition is administered suprachoroidally as compared to a level of AAV at the site of injection after the same pharmaceutical composition is administered via a subretinal administration or via an intravenous administration. In some embodiments, a level of AAV at the site of injection after the pharmaceutical composition is administered suprachoroidally is equal to or higher as compared to a level of AAV at the site of injection after a reference pharmaceutical composition is administered via a subretinal, or intravenous administration, or to the SCS. In some embodiments, the pharmaceutical composition has a higher level of AAV aggregation than the reference pharmaceutical composition. In some embodiments, the pharmaceutical composition and the reference pharmaceutical composition have the same vector genome concentration. In some embodiments, the pharmaceutical composition and a reference pharmaceutical composition have the same amount of genome copies. In some embodiments, the increase in the level of AAV at the site of injection is an increase of at least about 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%.

[0263] In some embodiments, the level of AAV at the site of injection after the pharmaceutical composition is administered to the SCS is increased by at least about 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500% as compared to the level of AAV at the site of injection after the same pharmaceutical composition is administered via subretinal or via intravitreous administrations. In some embodiments, the AAV level at the site of injection after the pharmaceutical composition is administered to the SCS is increased by at least about 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500% as compared to the AAV level at the site of injection after a reference pharmaceutical composition is administered to the SCS, or via subretinal, or via intravitreous administrations. In some embodiments, the pharmaceutical composition has a higher level of AAV aggregation as compared to the reference pharmaceutical composition.

[0264] In some embodiments, the AAV level or the rate of transduction (rate of infection) is determined about 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 15 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or at most about 400 days after administration.4.2.5 Transgene Expression

[0265] In some embodiments, the concentration of a transgene product is at least equal to or higher after a pharmaceutical composition is injected in the SCS as compared to after a reference pharmaceutical composition is injected in the SCS. In some embodiments, the concentration of a transgene product is at least equal to or higher after a pharmaceutical composition is injected in the SCS as compared to after a reference pharmaceutical composition is injected by subretinal injection or by intravitreous injection. In some embodiments, the concentration of a transgene product is at least equal to or higher after a pharmaceutical composition is injected in the SCS as compared to after the same pharmaceutical composition is injected by subretinal injection or by intravitreous injection.

[0266] In some embodiments, a transgene product (e.g., concentration of the transgene product) is detected in an eye (e.g., vitreous humor) for a longer period of time after a pharmaceutical composition is injected in the SCS as compared to after a reference pharmaceutical composition is injected in the SCS. In some embodiments, a transgene product (e.g., concentration of the transgene product) is detected in an eye (e.g., vitreous humor) for a longer period of time after a pharmaceutical composition is injected in the SCS as compared to after a reference pharmaceutical composition is injected by subretinal injection or by intravitreous administration. In some embodiments, a transgene product (e.g., concentration of the transgene product) is detected in an eye (e.g., vitreous humor) for a longer period of time after a pharmaceutical composition is injected in the SCS as compared to after the same pharmaceutical composition is injected by subretinal injection or by intravitreous injection.

[0267] In some embodiments, the longer period of time is at least about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days longer. In some embodiments, the longer period of time is about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days longer.

[0268] In some embodiments, the transgene is detected in an eye (e.g., vitreous humor) for period of time, after the pharmaceutical composition is administered in the SCS, that is at least about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days after the administration.

[0269] In some embodiments, the transgene is detected in an eye (e.g., vitreous humor) for a period of time (e.g., after the reference pharmaceutical composition is administered via subretinal administration or via intravitreous administration or to the SCS; or after the pharmaceutical composition is administered via subretinal or via intravitreous administration) that is at most about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, or 100 days after administration.

[0270] In some embodiments, the concentration of a transgene product in an eye (e.g., vitreous humor) can be determined 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days after the pharmaceutical composition or the reference pharmaceutical composition is administered.

[0271] In some embodiments, a suprachoroidal administration of a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in a higher concentration of the transgene that is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater than after a reference pharmaceutical composition is used, for example, to administer the AAV comprising the expression cassette encoding the transgene by suprachoroidal administration.

[0272] In some embodiments, a suprachoroidal administration of a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in a higher concentration of the transgene that is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater than when a reference pharmaceutical composition (comprising lower levels of AAV aggregation or no detectable AAV aggregation) is used, for example, to administer the AAV comprising the expression cassette encoding the transgene by subretinal administration or by intravitreous administration.

[0273] In some embodiments, a suprachoroidal administration of a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation comprising an AAV comprising an expression cassette encoding a transgene) results in a higher concentration of the transgene that is at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, at least 50 times greater, at least 100 times greater, at least 5% greater, at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40%, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 150% greater, or at least 200% greater, at least 250% greater, or at least 300%, at least 400% greater, or at least 500% greater than when the same pharmaceutical composition is administered via subretinal administration or via intravitreous administration.

[0274] In some embodiments, the concentration of the transgene product after a pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) is administered by suprachoroidal injection is greater than after a reference pharmaceutical composition is administered by suprachoroidal injection. In some embodiments, the concentration of the transgene product after a pharmaceutical composition (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) is administered by suprachoroidal injection is greater than after a reference pharmaceutical composition is administered by subretinal administration or via intravitreous administration,

[0275] In some embodiments, the pharmaceutical compositions disclosed herein (e.g., a pharmaceutical composition comprising an AAV comprising an expression cassette encoding a transgene) provide greater transgene expression and / or tissue / cell transduction at the back of the eye (e.g., retina) than in the outer layer of the eye (e.g., sclera) through SCS delivery. Such features of the presently disclosed pharmaceutical compositions are advantageous because subretinal delivery is not required to achieve higher expression of the ocular transgenes at the back of the eye than the outer layer of the eye by using the presently disclosed pharmaceutical compositions subretinally.

[0276] In some embodiments, the concentration of a transgene product (TP) is equal to or higher in the retina after a presently disclosed pharmaceutical composition comprising AAV encoding the TP is injected in the SCS than a reference pharmaceutical composition comprising the same AAV is injected in the SCS. In other embodiments, the concentration of a TP is equal to or higher in the retina and the concentration of the TP is lower in the sclera after a pharmaceutical composition comprising AAV encoding the TP is injected in the SCS as compared to a reference pharmaceutical composition comprising the same AAV is injected in the SCS.4.2.6 Other Functional Properties

[0277] In some embodiments, the pharmaceutical composition described herein has a desired level of AAV aggregation that is suitable for suprachoroidal injection. In some embodiments, the recombinant AAV in the pharmaceutical composition is at least as stable as the recombinant AAV in a reference pharmaceutical composition (or a comparable pharmaceutical composition). In some embodiments, the recombinant AAV in the pharmaceutical composition is at least 50% as stable as the recombinant AAV in a reference pharmaceutical composition (or a comparable pharmaceutical composition). In some embodiments, the recombinant AAV in the pharmaceutical composition has at least the same or a comparable infectivity level as the recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has at least the same or a comparable free DNA level as the recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has at least the same or a comparable in vitro relative potency (IVRP) as the recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has at least the same or a comparable change in size level as the recombinant AAV in a reference pharmaceutical composition.

[0278] In certain embodiments, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable to freeze / thaw cycles than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as stable to freeze / thaw cycles as the same recombinant AAV in a reference pharmaceutical composition. In certain embodiments, the stability of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6 and Section 5.

[0279] In certain embodiments, the recombinant AAV in the pharmaceutical composition exhibits at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more infectivity than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% the infectivity of the same recombinant AAV in a reference pharmaceutical composition. In certain embodiments, the virus infectivity of the recombinant AAV is determined by an assay or assays disclosed in the present disclosure. In certain embodiments, the size of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6 and Section 5. In certain embodiments, the size is measured prior to or after freeze / thaw cycles.

[0280] In certain embodiments, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable over a period of time (e.g., when stored at −20° C. or at 37° C.), for example, at least about or about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, about 4 years than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as stable over a period of time as the same recombinant AAV in a reference pharmaceutical composition. In certain embodiments, the stability over a period of time of the recombinant AAV is determined by an assay or assays disclosed in the present disclosure. In certain embodiments, the stability over a period of time of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6 and Section 5.

[0281] In certain embodiments, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 higher in vitro relative potency (IVRP) than the same recombinant AAV in a reference pharmaceutical composition (e.g., when stored at −20° C. or at 37° C.). In some embodiments, the recombinant AAV in the pharmaceutical composition has about the same in vitro relative potency (IVRP) as the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in vitro relative potency (IVRP) as the same recombinant AAV in a reference pharmaceutical composition. In certain embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by an assay or assays disclosed in the present disclosure. In certain embodiments, the in vitro relative potency (IVRP) is measured prior to or after freeze / thaw cycles. In certain embodiments, the in vitro relative potency (IVRP) of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6.

[0282] In certain embodiments, the recombinant AAV in the pharmaceutical composition has at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times less free DNA than the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has about the same amount of free DNA as the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has about not more than two times the amount of free DNA as the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% the amount of free DNA as the same recombinant AAV in a reference pharmaceutical composition. In some embodiments, the recombinant AAV in the pharmaceutical composition has at least about 50% more, about 25% more, about 15% more, about 10% more, about 5% more, about 4% more, about 3% more, about 2% more, about 1% more, about 0% more, about 1% less, about 2% less, about 5% less, about 7% less, about 10% less, about 2 times more, about 3 times more, about 2 times less, or about 3 times less free DNA than the same recombinant AAV in a reference pharmaceutical composition. In certain embodiments, the free DNA of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6 and Section 5.

[0283] In certain embodiments, the recombinant AAV in the pharmaceutical composition has at most 20%, 15%, 10%, 8%, 5%, 4%, 10%, 2%, or 1% change in size over a period of time (e.g., when stored at −20° C. or at 37° C.), for example, at least about or about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 24 months, about 2 years, about 3 years, and about 4 years. In certain embodiments, the size of the recombinant AAV is determined by an assay or assays disclosed in the present disclosure. In certain embodiments, the size is measured prior to or after freeze / thaw cycles. In certain embodiments, the size of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6.

[0284] In certain embodiments, the recombinant AAV in the pharmaceutical composition is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times more stable than the same recombinant AAV in a reference pharmaceutical composition (e.g., when stored at −20° C. or at 37° C.). In some embodiments, the recombinant AAV in the pharmaceutical composition is about as stable as the same recombinant AAV in a reference pharmaceutical composition (e.g., when stored at −20° C. or at 37° C.). In some embodiments, the recombinant AAV in the pharmaceutical composition is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as stable as the same recombinant AAV in a reference pharmaceutical composition (e.g., when stored at −20° C. or at 37° C.). In certain embodiments, the stability of the recombinant AAV is determined by an assay or assays disclosed in Section 4.6.

[0285] In certain embodiments, a pharmaceutical composition provided herein is capable of being stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months without loss of stability as determined, e.g. by an assay or assays disclosed in Section 4.6 or. In certain embodiments, a pharmaceutical composition provided herein is capable of being stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at 4° C. without loss of stability. In certain embodiments, a pharmaceutical composition provided herein is capable of being stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at ≤60° C. without loss of stability. In certain embodiments, a pharmaceutical composition provided herein is capable of being stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at −80° C. without loss of stability. In certain embodiments, a pharmaceutical composition provided herein is capable of being stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at 4° C. after having been stored at −20° C. for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months without loss of stability.

[0286] In certain embodiments, a pharmaceutical composition provided herein is capable of being first stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at −80° C., then being thawed and, after thawing, being stored at 2-10° C., 4-8° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C. or 9° C. for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 additional months without loss of stability as determined, e.g., by an assay or assays disclosed in Section 4.6 or. In certain embodiments, a pharmaceutical composition provided herein is capable of being first stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at −80° C., then being thawed and, after thawing, being stored at about 4° C. for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 additional months without loss of stability as determined, e.g., by an assay or assays disclosed in Section 4.6 or 5. In certain embodiments, a pharmaceutical composition provided herein is capable of being first stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months at ≤60° C., then being thawed and, after thawing, being stored at about 4° C. for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 additional months without loss of stability as determined, e.g., by an assay or assays disclosed in Section 4.6 or 5.

[0287] Effects of the methods or pharmaceutical compositions provided herein may be monitored by measuring signs of vision loss, infection, inflammation and other safety events, including retinal detachment. In some embodiments, different pharmaceutical compositions (e.g., diluted formulation or lower ionic strength formulation) having different AAV aggregation levels can be used to deliver the vector in the SCS. In some embodiments, vectors delivered using a pharmaceutical composition comprising aggregated AAV (e.g., diluted formulation or lower ionic strength formulation) are more effective than vectors delivered using a reference pharmaceutical composition (e.g., when administered in the SCS). In some embodiments, vectors delivered using a formulation comprising aggregated AAV results in improved vision as compared to vectors delivered using a formulation comprising lower levels of aggregated AAV or no detectable level of aggregated AAV.

[0288] Effects of the methods or pharmaceutical compositions provided herein may also be measured by a change from baseline in National Eye Institute Visual Functioning Questionnaire, the Rasch-scored version (NEI-VFQ-28-R) (composite score; activity limitation domain score; and socio-emotional functioning domain score). In some embodiments, effects of the methods provided herein may also be measured by a change from baseline in National Eye Institute Visual Functioning Questionnaire 25-item version (NEI-VFQ-25) (composite score and mental health subscale score). In some embodiments, effects of the methods provided herein may also be measured by a change from baseline in Macular Disease Treatment Satisfaction Questionnaire (MacTSQ) (composite score; safety, efficacy, and discomfort domain score; and information provision and convenience domain score).

[0289] In specific embodiments, the efficacy of a method or vector (vector formulation) described herein is reflected by an improvement in vision at about 4 weeks, 12 weeks, 6 months, 12 months, 24 months, 36 months, or at other desired timepoints. In a specific embodiment, the improvement in vision is characterized by an increase in BCVA, for example, an increase by 1 letter, 2 letters, 3 letters, 4 letters, 5 letters, 6 letters, 7 letters, 8 letters, 9 letters, 10 letters, 11 letters, or 12 letters, or more. In a specific embodiment, the improvement in vision is characterized by a 5%, 10%, 15%, 20%, 30%, 40%, 50% or more increase in visual acuity from baseline.

[0290] In specific embodiments, there is no inflammation in the eye after treatment or little inflammation in the eye after treatment (for example, an increase in the level of inflammation by 10%, 5%, 2%, 1% or less from baseline).4.2.7 Reduction of AAV Empty Particles and Capsids

[0291] In some embodiments, provided herein is a pharmaceutical composition of the disclosure comprising a recombinant AAV vector comprising an expression cassette encoding a transgene, and comprising low or undetectable levels of AAV empty capsids or AAV empty particles. In some embodiments, a pharmaceutical composition of the disclosure comprises lower amounts of AAV empty capsids or AAV empty particles as compared to a reference pharmaceutical composition. In some embodiments, the amount of the AAV empty capsids or AAV empty particles in the pharmaceutical composition is lower by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the amount of the AAV empty capsids or AAV empty particles in a reference pharmaceutical composition. In some embodiments, the amount of the AAV empty capsids or AAV empty particles in the pharmaceutical composition is lower by about or at least about 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 6 folds, 7 folds, 8 folds, 9 folds, 10 folds, 15 folds, 20 folds, 25 folds, 30 folds, 40 folds, 45 folds, 50 folds, 55 folds, 60 folds, 65 folds, 70 folds, 75 folds, 80 folds, 85 folds, 90 folds, 95 folds, 100 folds, or more than 100 folds as compared to the amount of the AAV empty capsids or AAV empty particles in a reference pharmaceutical composition. In some embodiments, the pharmaceutical composition of the disclosure comprising reduced or undetectable AAV empty capsids or AAV empty particles comprises an ionic strength of about or at most about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, or 60 mM. In some embodiments, the reference pharmaceutical composition comprises an ionic strength of more than about 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, or more than about 200 mM.

[0292] In some embodiments, disclosed herein is a method of reducing or eliminating AAV empty capsids or AAV empty particles. In some embodiments, the method comprises introducing a solution comprising an ionic strength of at most about 60 mM to a formulation comprising a mixture of a recombinant adeno-associated virus (AAV) vector of the disclosure and AAV empty capsids or AAV empty particles. In some embodiments, introducing a solution comprising an ionic strength of at most about 60 mM causes the rAAV of the disclosure to aggregate. In some embodiments, introducing a solution comprising an ionic strength of at most about 60 mM does not result in aggregation of the AAV empty capsids or AAV empty particles. In some embodiments, the method comprises removing the AAV empty capsids or AAV empty particles from the formulation after the solution comprising an ionic strength of at most about 60 mM is added to the formulation. In some embodiments, the formulation is then prepared into a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises the recombinant adeno-associated virus (AAV) vector, wherein the recombinant AAV vector comprises an expression cassette encoding a transgene, and wherein the pharmaceutical composition is suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject. In some embodiments, the method further comprises introducing another solution comprising an ionic strength of at least about 80 mM to the formulation after the empty AAV capsids or empty AAV particles are removed from the formulation. In some embodiments, the another solution comprises an ionic strength of about or at least about 150 mM.

[0293] In some embodiments, disclosed herein is a method of reducing or eliminating AAV empty capsids or AAV empty particles in a population of AAV particles. In some embodiments, the population of AAV particles comprises empty AAV particles and AAV particles comprising an expression cassette encoding a transgene. In some embodiments, the method comprises incubating the population of AAV particles in a solution at an ionic strength of at most about 60 mM. In some embodiments, incubating the population of AAV particles in a low ionic strength solution (e.g., about or less than about 60 mM) causes the AAV particles comprising the expression cassette encoding the transgene to aggregate while the empty AAV particles or empty AAV capsids remain unaggregated. In some embodiments, the method comprises removing at least a portion of the empty AAV particles from the population of AAV particles. In some embodiments, the method further comprises introducing another solution comprising an ionic strength of at least about 80 mM to the formulation after the empty AAV capsids or empty AAV particles are removed from the formulation. In some embodiments, the another solution comprises an ionic strength of about or at least about 150 mM.

[0294] In some embodiments, the amount of the AAV empty capsids or AAV empty particles in the formulation is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 95%, 96%, 97%, 98%, 99%, or 100% after the AAV empty capsids or AAV empty particles are removed from the formulation or solution as compared to the amount of the AAV empty capsids or AAV empty particles in the formulation or solution prior to adding a solution comprising low ionic strength (e.g., a solution comprising at most about 60 mM). In some embodiments, the solution or solution comprising low ionic strength is a solution comprising an ionic strength of about 30 to about 50 mM. In some embodiments, the solution or solution comprising low ionic strength is a solution comprising an ionic strength of about 15 to about 50 mM. In some embodiments, the solution or solution comprising low ionic strength is a solution comprising an ionic strength of about or at most about 50 mM.

[0295] In some embodiments, the pharmaceutical composition is free or substantially free of AAV empty capsids or AAV empty particles. The term “free of AAV empty capsids or AAV empty particles” refers to a pharmaceutical composition or formulation in which the level of AAV empty capsids or AAV empty particles is undetectable by a conventional method or an available method. The term “substantially free of AAV empty capsids or AAV empty particles” refers to a pharmaceutical composition or formulation in which the level of AAV empty capsids or AAV empty particles is at most about 5% of the AAV particles in a composition or solution.4.3 Dosage and Mode of Administration

[0296] In one aspect, provided herein is a method of suprachoroidal administration for treating a pathology of the eye, comprising administering to the suprachoroidal space in the eye of a human subject in need of treatment a recombinant viral vector comprising a nucleotide sequence encoding a therapeutic product such that the therapeutic product is expressed and results in treatment of the pathology of the eye. In certain embodiments, the administering step is by injecting the recombinant viral vector into the suprachoroidal space using a suprachoroidal drug delivery device. In certain embodiments, the suprachoroidal drug delivery device is a microinjector. In some embodiments, a pharmaceutical composition provided herein is suitable for administration by one, two or more routes of administration (e.g., suitable for suprachoroidal and subretinal administration).

[0297] In certain embodiments, the vector genome concentration (VGC) of the pharmaceutical composition (or the reference pharmaceutical composition) is about 3×109 GC / mL, about 1×1010 GC / mL, about 1.2×1010 GC / mL, about 1.6×1010 GC / mL, about 4×1010 GC / mL, about 6×1010 GC / mL, about 2×1011 GC / mL, about 2.4×1011 GC / mL, about 2.5×1011 GC / mL, about 3×1011 GC / mL, about 3.2×1011 GC / mL, about 6.2×1011 GC / mL, about 6.5×1011 GC / mL, about 1×1012 GC / mL, about 2.5×1012 GC / mL, about 3×1012 GC / mL, about 5×1012 GC / mL, about 1.5×1013 GC / mL, about 2×1013 GC / mL or about 3×1013 GC / mL.

[0298] In certain embodiments, the vector genome concentration (VGC) of the pharmaceutical composition (or the reference pharmaceutical composition) is about 3×109 GC / mL, 4×109 GC / mL, 5×109 GC / mL, 6×109 GC / mL, 7×109 GC / mL, 8×109 GC / mL, 9×109 GC / mL, about 1×1010 GC / mL, about 2×1010 GC / mL, about 3×1010 GC / mL, about 4×1010 GC / mL, about 5×1010 GC / mL, about 6×1010 GC / mL, about 7×1010 GC / mL, about 8×1010 GC / mL, about 9×1010 GC / mL, about 1×1011 GC / mL, about 2×1011 GC / mL, about 3×1011 GC / mL, about 4×1011 GC / mL, about 5×1011 GC / mL, about 6×1011 GC / mL, about 7×1011 GC / mL, about 8×1011 GC / mL, about 9×1011 GC / mL, about 1×1012 GC / mL, about 2×1012 GC / mL, about 3×102 GC / mL, about 4×1012 GC / mL, about 5×1012 GC / mL, about 6×1012 GC / mL, about 7×1012 GC / mL, about 8×1012 GC / mL, about 9×1012 GC / mL, about 1×1013 GC / mL, about 1.5×1013 GC / mL, about 2×1013 GC / mL, about 3×1013 GC / mL.

[0299] In some embodiments, the volume of the pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) is any volume capable of reducing the minimum force to separate the sclera and choroid. In some embodiments, the volume of the pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) is about 50 μL to about 1000 μL, 50 μL to about 500 μL, 50 μL to about 400 μL, 50 μL to about 350 μL, 50 μL to about 300 μL, about 50 μL to about 275 μL, about 50 μL to about 250 μL, about 50 μL to about 225 μL, about 50 μL to about 200 μL, about 50 μL to about 175 μL, about 50 μL to about 150 μL, about 60 μL to about 140 μL, about 70 μL to about 130 μL, about 80 μL to about 120 μL, about 90 μL to about 110 μL, or about 100 μL.

[0300] Currently available technologies for suprachoroidal space (SCS) delivery exist. Preclinically, SC injections have been achieved with scleral flap technique, catheters and standard hypodermic needles, as well as with microneedles. A hollow-bore 750 μm-long microneedle (Clearside Biomedical, Inc.) can be inserted at the pars, and has shown promise in clinical trials. A microneedle designed with force-sensing technology can be utilized for SC injections, as described by Chitnis, et al. (Chitnis, G. D., et al. A resistance-sensing mechanical injector for the precise delivery of liquids to target tissue. Nat Biomed Eng 3, 621-631 (2019). https: / / doi.org / 10.1038 / s41551-019-0350-2). Oxular Limited is developing a delivery system (Oxulumis) that advances an illuminated cannula in the suprachoroidal space. The Orbit device (Gyroscope) is a specially-designed system enabling cannulation of the suprachoroidal space with a flexible cannula. A microneedle inside the cannula is advanced into the subretinal space to enable targeted dose delivery. Ab interno access to the SCS can also be achieved using micro-stents, which serve as minimally-invasive glaucoma surgery (MIGS) devices. Examples include the CyPass® Micro-Stent (Alcon, Fort Worth, Texas, US) and iStent® (Glaukos), which are surgically implanted to provide a conduit from the anterior chamber to the SCS to drain the aqueous humor without forming a filtering bleb. Other devices contemplated for suprachoroidal delivery include those described in UK Patent Publication No. GB 2531910A and U.S. Pat. No. 10,912,883 B2.

[0301] In some embodiments, the suprachoroidal drug delivery device is a syringe with a 1 millimeter 30 gauge needle. In some embodiments, the syringe has a larger circumference (e.g., 29 gauge needle). During an injection using this device, the needle pierces to the base of the sclera and fluid containing drug enters the suprachoroidal space, leading to expansion of the suprachoroidal space. As a result, there is tactile and visual feedback during the injection. Following the injection, the fluid flows posteriorly and absorbs dominantly in the choroid and retina. This results in the production of transgene protein from all retinal cell layers and choroidal cells. Using this type of device and procedure allows for a quick and easy in-office procedure with low risk of complications.

[0302] In some embodiments, a microneedle or syringe is selected based on the level of AAV aggregation of a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation). In some embodiments, a microneedle is selected based on the pressure resulted in the eye (e.g., in the SCS) when a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) is administered. For example, a pharmaceutical composition (e.g., diluted formulation or lower ionic strength formulation) having higher levels of AAV aggregation may benefit from the use of a wider microneedle for injection. In some embodiments, the pressure in the SCS is lower when a wider microneedle is used as compared to the pressure obtained when a narrower microneedle is used. In some embodiments, 10 gauge needle, 11 gauge needle, 12 gauge needle, 13 gauge needle, 14 gauge needle, 15 gauge needle, 16 gauge needle, 17 gauge needle, 18 gauge needle, 19 gauge needle, 20 gauge needle, 21 gauge needle, 22 gauge needle, 23 gauge needle, 24 gauge needle, 25 gauge needle, 26 gauge needle, 27 gauge needle, 28 gauge needle, 29 gauge needle, 30 gauge needle, 31 gauge needle, 32 gauge needle, 33 gauge needle, or 34 gauge needle is used. In some embodiments, a 27 gauge needle is used. In some embodiments, a 28 gauge needle is used. In some embodiments, a 29 gauge needle is used. In some embodiments, a 30 gauge needle is used. In some embodiments, a 31 gauge needle is used. In some embodiments, a gauge that is smaller than a 27 gauge needle is used. In some embodiments, a gauge that is larger than a 27 gauge needle is used. In some embodiments, a gauge that is smaller than a 30 gauge needle is used. In some embodiments, a gauge that is higher than a 30 gauge needle is used.

[0303] In some embodiments, the pressure during administration of a pharmaceutical composition is about 10 PSI, 15 PSI, 20 PSI, 25 PSI, 30 PSI, 35 PSI, 40 PSI, 45 PSI, 50 PSI, 55 PSI, 60 PSI, 65 PSI, 70 PSI, 75 PSI, 80 PSI, 85 PSI, 90 PSI, 95 PSI, 100 PSI, 150 PSI, or 200 PSI. In some embodiments, the pressure during administration of a pharmaceutical composition is not greater than about 10 PSI, 15 PSI, 20 PSI, 25 PSI, 30 PSI, 35 PSI, 40 PSI, 45 PSI, 50 PSI, 55 PSI, 60 PSI, 65 PSI, 70 PSI, 75 PSI, 80 PSI, 85 PSI, 90 PSI, 95 PSI, 100 PSI, 150 PSI, or 200 PSI. In some embodiments, the pressure to open the SCS during administration of a pharmaceutical composition is not greater than about 10 PSI, 15 PSI, 20 PSI, 25 PSI, 30 PSI, 35 PSI, 40 PSI, 45 PSI, 50 PSI, 55 PSI, 60 PSI, 65 PSI, 70 PSI, 75 PSI, 80 PSI, 85 PSI, 90 PSI, 95 PSI, 100 PSI, 150 PSI, or 200 PSI. In some embodiments, the pressure during administration of a pharmaceutical composition (or the pressure required to open the SCS) is between 20 PSI and 50 PSI, 20 PSI and 75 PSI, 20 PSI and 40 PSI, 10 PSI and 40 PSI, 10 PSI and 100 PSI, or 10 PSI and 80 PSI. In some embodiments, the pressure decreases as the rate of injection decreases (e.g., pressure decreases from a 4 seconds rate of injection to a 10 seconds rate of injection). In some embodiments, the pressure decreases as the size of the needle increases. In some embodiments, the pressure increases as the level of AAV aggregation increases.

[0304] Doses that maintain a concentration of the transgene product at a Cmin of at least 0.330 μg / mL in the eye (e.g., Vitreous humor), or 0.110 μg / mL in the Aqueous humour (the anterior chamber of the eye) for three months are desired; thereafter, Vitreous Cmin concentrations of the transgene product ranging from 1.70 to 6.60 μg / mL, and / or Aqueous Cmin concentrations ranging from 0.567 to 2.20 μg / mL should be maintained. However, because the transgene product is continuously produced (under the control of a constitutive promoter or induced by hypoxic conditions when using an hypoxia-inducible promoter), maintenance of lower concentrations can be effective. Transgene concentrations can be measured directly in patient samples of fluid collected from a bodily fluid, ocular fluid, vitreous humor, or the anterior chamber, or estimated and / or monitored by measuring the patient's serum concentrations of the transgene product—the ratio of systemic to vitreal exposure to the transgene product is about 1:90,000. (E.g., see, vitreous humor and serum concentrations reported in Xu L, et al., 2013, Invest. Opthal. Vis. Sci. 54: 1616-1624, at p. 1621 and Table 5 at p. 1623, which is incorporated by reference herein in its entirety).

[0305] In certain embodiments, dosages are measured by genome copies per ml (GC / mL) or the number of genome copies administered to the eye of the patient (e.g., administered suprachoroidally). In some embodiments, 2.4×1011 GC / mL to 1×1013 GC / mL are administered, 2.4×1011 GC / mL to 5×1011 GC / mL are administered, 5×1011 GC / mL to 1×1012 GC / mL are administered, 1×1012 GC / mL to 5×1012 GC / mL are administered, or 5×1012 GC / mL to 1×1013 GC / mL are administered. In some embodiments, 1.5×1013 GC / mL to 3×1013 GC / mL are administered. In some embodiments, about 2.4×1011 GC / mL, about 5×1011 GC / mL, about 1×1012 GC / mL, about 2.5×1012 GC / mL, about 5×1012 GC / mL, about 1×1013 GC / mL or about 1.5×1013 GC / mL are administered. In some embodiments, 1×109 to 1×1012 genome copies are administered. In some embodiments, 3×109 to 2.5×1011 genome copies are administered. In specific embodiments, 1×109 to 2.5×1011 genome copies are administered. In specific embodiments, 1×109 to 1×1011 genome copies are administered. In specific embodiments, 1×109 to 5×109 genome copies are administered. In specific embodiments, 6×109 to 3×1010 genome copies are administered. In specific embodiments, 4×1010 to 1×1011 genome copies are administered. In specific embodiments, 2×1011 to 1×1012 genome copies are administered. In a specific embodiment, about 3×109 genome copies are administered (which corresponds to about 1.2×1010 GC / mL in a volume of 250 μl). In another specific embodiment, about 1×1010 genome copies are administered (which corresponds to about 4×1010 GC / mL in a volume of 250 μl). In another specific embodiment, about 6×1010 genome copies are administered (which corresponds to about 2.4×1011 GC / mL in a volume of 250 μl). In another specific embodiment, about 6.4×1010 genome copies are administered (which corresponds to about 3.2×1011 GC / mL in a volume of 200 μl). In another specific embodiment, about 1.3×1011 genome copies are administered (which corresponds to about 6.5×1011 GC / mL in a volume of 200 μl). In another specific embodiment, about 2.5×1011 genome copies are administered (which corresponds to about 2.5×1012 GC / mL in a volume of 100 μl). In another specific embodiment, about 5×1011 genome copies are administered (which corresponds to about 5×1012 GC / mL in a volume of 200 μl). In another specific embodiment, about 1.5×1012 genome copies are administered (which corresponds to about 1.5×1013 GC / mL in a volume of 100 μl). In some embodiments, about 6.4×1010 genome copies are administered per eye, or per dose, or per route of administration. In some embodiments, about 6.4×1010 genome copies is the total number of genome copies administered. In some embodiments, about 1.3×1011 genome copies are administered per eye, or per dose, or per route of administration. In some embodiments, about 1.3×1011 genome copies is the total number of genome copies administered. In some embodiments, about 2.5×1011 genome copies are administered per eye, or per dose, or per route of administration. In some embodiments, about 2.5×1011 genome copies is the total number of genome copies administered. In some embodiments, about 5×1011 genome copies are administered per eye, or per dose, or per route of administration. In some embodiments, about 5×1011 genome copies is the total number of genome copies administered. In some embodiments, about 1.5×1012 genome copies are administered per eye, or per dose, or per route of administration. In some embodiments, about 1.5×1012 genome copies is the total number of genome copies administered. In some embodiments, about 3×1012 genome copies are administered per eye, or per dose, or per route of administration. In some embodiments, about 3×1012 genome copies is the total number of genome copies administered. In another specific embodiment, about 1.6×1011 genome copies are administered (which corresponds to about 6.2×1011 GC / mL in a volume of 250 μl). In another specific embodiment, about 1.55×1011 genome copies are administered (which corresponds to about 6.2×1011 GC / mL in a volume of 250 μl). In another specific embodiment, about 1.6×1011 genome copies are administered (which corresponds to about 6.4×1011 GC / mL in a volume of 250 μl). In another specific embodiment, about 2.5×1011 genome copies (which corresponds to about 1.0×1012 in a volume of 250 μl) are administered. In another specific embodiment, about 3×1011 genome copies are administered (which corresponds to about 3×1012 GC / mL in a volume of 100 μl). In another specific embodiment, about 6×1011 genome copies are administered (which corresponds to about 3×1012 GC / mL in a volume of 200 μl). In another specific embodiment, about 6×1011 genome copies are administered (which corresponds to about 6×1012 GC / mL in a volume of 100 μl).

[0306] In certain embodiments, about 6.0×1010 genome copies per administration, or per eye are administered. In certain embodiments, about 6.4×1010 genome copies per administration, or per eye are administered. In certain embodiments, about 1.3×1011 genome copies per administration, or per eye are administered. In certain embodiments, about 1.5×1011 genome copies per administration, or per eye are administered. In certain embodiments, about 1.6×1011 genome copies per administration, or per eye are administered. In certain embodiments, about 2.5×1011 genome copies per administration, or per eye are administered. In certain embodiments, about 3×1011 genome copies per administration, or per eye are administered. In certain embodiments, about 5.0×1011 genome copies per administration, or per eye are administered. In certain embodiments, about 6×1011 genome copies per administration, or per eye are administered. In certain embodiments, about 3×1012 genome copies per administration, or per eye are administered. In certain embodiments, about 1.0×1012 GC / mL per administration, or per eye are administered. In certain embodiments, about 2.5×1012 GC / mL per administration, or per eye are administered. In certain embodiments, about 3×1012 GC / mL per administration, or per eye are administered. In certain embodiments, about 3.0×1013 genome copies per administration, or per eye are administered. In certain embodiments, up to 3.0×1013 genome copies per administration, or per eye are administered.

[0307] In certain embodiments, about 1.5×1011 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 2.5×1011 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 3×1011 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 5.0×1011 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 6×1011 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 1.5×1012 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 3×1012 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 2.5×1011 genome copies per eye are administered by a single suprachoroidal injection. In certain embodiments, about 3×1011 genome copies per administration, or per eye are administered by a single suprachoroidal injection. In certain embodiments, about 3×1011 genome copies per administration, or per eye are administered by a single suprachoroidal injection in a volume of 100 μl. In certain embodiments, about 3×1011 genome copies per administration, or per eye are administered by a single suprachoroidal injection in a volume of 200 μl. In certain embodiments, about 3×1011 genome copies per administration, or per eye are administered by double suprachoroidal injections. In certain embodiments, about 3×1011 genome copies per administration, or per eye are administered by double suprachoroidal injections, wherein each injection is in a volume of 50 μl. In certain embodiments, about 3×1011 genome copies per administration, or per eye are administered by double suprachoroidal injections, wherein each injection is in a volume of 100 μl. In certain embodiments, about 5.0×1011 genome copies per administration, or per eye are administered by double suprachoroidal injections. In certain embodiments, about 6×1011 genome copies per administration, or per eye are administered by a single suprachoroidal injection. In certain embodiments, about 6×1011 genome copies per administration, or per eye are administered by a single suprachoroidal injection in a volume of 100 μl. In certain embodiments, about 6×1011 genome copies per administration, or per eye are administered by a single suprachoroidal injection in a volume of 200 μl. In certain embodiments, about 6×1011 genome copies per administration, or per eye are administered by double suprachoroidal injections. In certain embodiments, about 6×1011 genome copies per administration, or per eye are administered by double suprachoroidal injections, wherein each injection is in a volume of 50 μl. In certain embodiments, about 6×1011 genome copies per administration, or per eye are administered by double suprachoroidal injections, wherein each injection is in a volume of 100 μl. In certain embodiments, about 3.0×1013 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, up to 3.0×1013 genome copies per administration, or per eye are administered by suprachoroidal injection. In certain embodiments, about 2.5×1012 GC / mL per eye are administered by a single suprachoroidal injection in a volume of 100 μl. In certain embodiments, about 2.5×1012 GC / mL per eye are administered by double suprachoroidal injections, wherein each injection is in a volume of 100 μl. In certain embodiments, about 1.5×1013 GC / mL per eye are administered by a single suprachoroidal injection in a volume of 100 μl.

[0308] In certain embodiments, the recombinant viral vector is administered by double suprachoroidal injections. In certain embodiments, the first injection in the right eye is administered in the superior temporal quadrant (i.e., between the 10 o'clock and 11 o'clock positions), and the second injection in the same eye is administered in the inferior nasal quadrant (i.e., between the 4 o'clock and 5 o'clock positions). In certain embodiments, the first injection in the right eye is administered in the inferior nasal quadrant (i.e., between the 4 o'clock and 5 o'clock positions), and the second injection in the same eye is administered in the superior temporal quadrant (i.e., between the 10 o'clock and 11 o'clock positions). In certain embodiments, the first injection in the left eye is administered in the superior temporal quadrant (i.e., between the 1 o'clock and 2 o'clock positions), and the second injection in the same eye is administered in the inferior nasal quadrant (i.e., between the 7 o'clock and 8 o'clock positions). In certain embodiments, the first injection in the left eye is administered in the inferior nasal quadrant (i.e., between the 7 o'clock and 8 o'clock positions), and the second injection in the same eye is administered in the superior temporal quadrant (i.e., between the 1 o'clock and 2 o'clock positions).

[0309] In certain embodiments, the recombinant viral vector is administered by a single suprachoroidal injection. In certain embodiments, the single injection in the right eye is administered in the superior temporal quadrant (i.e., between the 10 o'clock and 11 o'clock positions). In certain embodiments, the single injection in the right eye is administered in the inferior nasal quadrant (i.e., between the 4 o'clock and 5 o'clock positions). In certain embodiments, the single injection in the left eye is administered in the superior temporal quadrant (i.e., between the 1 o'clock and 2 o'clock positions). In certain embodiments, the single injection in the left eye is administered in the inferior nasal quadrant (i.e., between the 7 o'clock and 8 o'clock positions).

[0310] In some embodiments, the pharmaceutical composition or the reference pharmaceutical composition is administered to a human subject (e.g., suprachoroidally, subretinally, or intravitreously) once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, twenty-five times, or thirty times. In some embodiments, the pharmaceutical composition or the reference pharmaceutical composition is administered to a human subject once in one day, twice in one day, three times in one day, four times in one day, five times in one day, six times in one day, or seven times in one day. In some embodiments, the same amount of AAV genome copies are administered per administration. For example, the same genome copies are administered suprachoroidally, subretinally, or intravitreously. In some embodiments, the same total amount of AAV genome copies are administered. For example, the same total amount of AAV genome copies are administered suprachoroidally, subretinally, or intravitreously regardless of the number of total administrations (e.g., if subretinal administration is performed once and suprachoroidal administration is performed twice, the genome copies in the one subretinal administration is the same as the genome copies in both suprachoroidal administrations combined).

[0311] As used herein and unless otherwise specified, the term “about” means within plus or minus 10% of a given value or range4.4 Constructs and Formulations

[0312] In some embodiments, the recombinant vectors provided herein comprise the following elements in the following order: a) a constitutive or a hypoxia-inducible promoter sequence, and b) a sequence encoding the transgene (e.g., therapeutic product). In certain embodiments, the recombinant vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or a hypoxia-inducible promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding the transgene, i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, l) a fifth linker sequence, and m) a second ITR sequence.

[0313] In certain embodiments, the recombinant vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or a hypoxia-inducible promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding the transgene, i) a second UTR sequence, j) a fourth linker sequence, k) a poly A sequence, l) a fifth linker sequence, and m) a second ITR sequence, wherein the transgene encodes a light chain and a heavy chain sequence separated by a cleavable F / F2A sequence.

[0314] In some embodiments, the AAV (AAV viral vectors) provided herein comprise the following elements in the following order: a) a constitutive or a hypoxia-inducible promoter sequence, and b) a sequence encoding the transgene. In some embodiments, the AAV used for delivering the transgene should have a tropism for human retinal cells or photoreceptor cells. Such AAV can include non-replicating recombinant adeno-associated virus vectors (“rAAV”), particularly those bearing an AAV8 capsid are preferred. In some embodiments, the viral vector comprises a signal peptide. In some embodiments, the signal peptide is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 55). In some embodiments, the signal peptide is derived from IL-2 signal sequence. In some embodiments, the viral vector comprises a signal peptide from any signal peptide disclosed in Table 1, such as MNFLLSWVHW SLALLLYLHH AKWSQA (VEGF-A signal peptide) (SEQ ID NO: 5); MERAAPSRRV PLPLLLLGGL ALLAAGVDA (Fibulin-1 signal peptide) (SEQ ID NO: 6); MAPLRPLLIL ALLAWVALA (Vitronectin signal peptide) (SEQ ID NO: 7); MRLLAKIICLMLWAICVA (Complement Factor H signal peptide) (SEQ ID NO: 8); MRLLAFLSLL ALVLQETGT (Opticin signal peptide) (SEQ ID NO: 9); MKWVTFISLLFLFSSAYS (Albumin signal peptide) (SEQ ID NO: 22); MAFLWLLSCWALLGTTFG (Chymotrypsinogen signal peptide) (SEQ ID NO: 23); MYRMQLLSCIALILALVTNS (Interleukin-2 signal peptide) (SEQ ID NO: 24); MNLLLILTFVAAAVA (Trypsinogen-2 signal peptide) (SEQ ID NO: 25); or MYRMQLLLLIALSLALVTNS (mutant Interleukin-2 signal peptide) (SEQ ID NO: 55).

[0315] In some embodiments, the viral vector or other expression construct suitable for packaging in an AAV capsid, comprises (1) AAV inverted terminal repeats (ITRs) flank the expression cassette; (2) regulatory control elements, consisting essentially of one or more enhancers and / or promoters, d) a poly A signal, and e) optionally an intron; and (3) a transgene providing (e.g., coding for) one or more RNA or protein products of interest.

[0316] In some aspects, the disclosure provides for a nucleic acid for use, wherein the nucleic acid encodes a therapeutic product operatively linked to a promoter or enhancer-promoter described herein.

[0317] In some aspects, the disclosure provides for a nucleic acid for use, wherein the nucleic acid encodes a transgene operatively linked to a promoter selected from the group consisting of: the CB7 promoter (a chicken β-actin promoter and CMV enhancer), cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65 promoter and opsin promoter. In a specific embodiment, the transgene is operatively linked to the CB7 promoter.

[0318] In certain embodiments, provided herein are recombinant vectors that comprise one or more nucleic acids (e.g. polynucleotides). The nucleic acids may comprise DNA, RNA, or a combination of DNA and RNA. In certain embodiments, the DNA comprises one or more of the sequences selected from the group consisting of promoter sequences, the sequence encoding the therapeutic product of interest (the transgene), untranslated regions, and termination sequences. In certain embodiments, recombinant vectors provided herein comprise a promoter operably linked to the sequence encoding the therapeutic product of interest.

[0319] In certain embodiments, nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59:149-161).

[0320] In certain embodiments, the recombinant vectors provided herein comprise modified mRNA encoding for the therapeutic product of interest (e.g., the transgene). In certain embodiments, the recombinant vectors provided herein comprise a nucleotide sequence encoding for a therapeutic product that is an shRNA, siRNA, or miRNA.

[0321] In certain embodiments, the vectors provided herein comprise components that modulate protein delivery. In certain embodiments, the viral vectors provided herein comprise one or more signal peptides. Examples of signal peptides include, but is not limited to, VEGF-A signal peptide (SEQ ID NO: 5), fibulin-1 signal peptide (SEQ ID NO: 6), vitronectin signal peptide (SEQ ID NO: 7), complement Factor H signal peptide (SEQ ID NO: 8), opticin signal peptide (SEQ ID NO: 9), albumin signal peptide (SEQ ID NO: 22), chymotrypsinogen signal peptide (SEQ ID NO: 23), interleukin-2 signal peptide (SEQ ID NO: 24), and trypsinogen-2 signal peptide (SEQ ID NO: 25), mutant interleukin-2 signal peptide (SEQ ID NO: 55).(a) Viral Vectors

[0322] In some embodiments, the viral vectors provided herein are AAV based viral vectors. In preferred embodiments, the viral vectors provided herein are AAV8 based viral vectors. In certain embodiments, the AAV8 based viral vectors provided herein retain tropism for retinal cells. In certain embodiments, the AAV-based vectors provided herein encode the AAV rep gene (required for replication) and / or the AAV cap gene (required for synthesis of the capsid proteins). Multiple AAV serotypes have been identified. In certain embodiments, AAV-based vectors provided herein comprise components from one or more serotypes of AAV. In certain embodiments, AAV based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHIM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, rAAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In preferred embodiments, AAV based vectors provided herein comprise components from one or more of AAV8, AAV9, AAV10, AAV11, or AAVrh10 serotypes. In certain embodiments, the recombinant viral vectors provided herein are altered such that they are replication-deficient in humans. In certain embodiments, the recombinant viral vectors are hybrid vectors, e.g., an AAV vector placed into a “helpless” adenoviral vector. In certain embodiments, provided herein are recombinant viral vectors comprising a viral capsid from a first virus and viral envelope proteins from a second virus. In specific embodiments, the second virus is vesicular stomatitis virus (VSV). In more specific embodiments, the envelope protein is VSV-G protein.

[0323] Provided in particular embodiments are AAV8 vectors comprising a viral genome comprising an expression cassette for expression of the transgene, under the control of regulatory elements and flanked by ITRs and a viral capsid that has the amino acid sequence of the AAV8 capsid protein or is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAV8 capsid protein (SEQ ID NO: 48) while retaining the biological function of the AAV8 capsid. In certain embodiments, the encoded AAV8 capsid has the sequence of SEQ ID NO: 48 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions and retaining the biological function of the AAV8 capsid.

[0324] In certain embodiments, the AAV that is used in the methods described herein is Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety. In certain embodiments, the AAV that is used in the methods described herein comprises one of the following amino acid insertions: LGETTRP or LALGETTRP, as described in U.S. Pat. Nos. 9,193,956; 9,458,517; and 9,587,282 and US patent application publication no. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV that is used in the methods described herein is AAV.7m8, as described in U.S. Pat. Nos. 9,193,956; 9,458,517; and 9,587,282 and US patent application publication no. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV that is used in the methods described herein is any AAV disclosed in U.S. Pat. No. 9,585,971, such as AAV.PHP.B. In certain embodiments, the AAV that is used in the methods described herein is an AAV disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: U.S. Pat. Nos. 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9,458,517; and 9,587,282 US patent application publication nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335.

[0325] AAV8-based viral vectors are used in certain of the methods described herein. Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in U.S. Pat. Nos. 7,282,199 B2, 7,790,449 B2, 8,318,480 B2, 8,962,332 B2 and International Patent Application No. PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety. In one aspect, provided herein are AAV (e.g., AAV8)-based viral vectors encoding a transgene.

[0326] In some embodiments, recombinant AAV viral vectors have been shown to have particular tropism for ocular tissues and are efficient at transducing and expressing the transgene product of such recombinant AAV. Thus, ocular-tropic vectors are useful in the methods and pharmaceutical compositions disclosed herein. In some embodiments, the method or pharmaceutical composition disclosed herein comprises an AAV viral vector having enhanced tropism for posterior segments of the eye, such as retina and RPE / choroid. In some embodiments, the method or pharmaceutical composition disclosed herein comprises a recombinant AAV3B viral vector. In certain embodiments, the AAV vector is an AAV vector disclosed in PCT International Application No. PCT / US2021 / 054008 (PCT International Publication No. WO2022076711A2, published Apr. 14, 2022), which is incorporated herein by reference in its entirety.

[0327] In certain embodiments, a single-stranded AAV (ssAAV) may be used supra. In certain embodiments, a self-complementary vector, e.g., scAAV, may be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82, McCarty et al, 2001, Gene Therapy, Vol 8, Number 16, Pages 1248-1254; and U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).

[0328] In certain embodiments, the viral vectors used in the methods described herein are adenovirus based viral vectors. A recombinant adenovirus vector may be used to transfer in the transgene. The recombinant adenovirus can be a first generation vector, with an E1 deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region. The recombinant adenovirus can be a second generation vector, which contains full or partial deletions of the E2 and E4 regions. A helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi). The transgene is inserted between the packaging signal and the 3′ITR, with or without stuffer sequences to keep the genome close to wild-type size of approx. 36 kb. An exemplary protocol for production of adenoviral vectors may be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,” Gene Therapy 12:S18-S27, which is incorporated by reference herein in its entirety.

[0329] In a specific embodiment, a vector for use in the methods described herein is one that encodes transgene such that, upon introduction of the vector into a relevant cell (e.g., a retinal cell in vivo or in vitro), a glycosylated and or tyrosine sulfated variant of the transgene product is expressed by the cell. In a specific embodiment, the expressed transgene product comprises a glycosylation and / or tyrosine sulfation pattern.(b) Therapeutic Product or Transgenes

[0330] The therapeutic products can be, for example, therapeutic proteins (for example, antibodies), therapeutic RNAs (for example, shRNAs, siRNAs, and miRNAs), or therapeutic aptamers.

[0331] In certain embodiments, the disclosure provides a pharmaceutical composition comprising recombinant AAV encoding a transgene. In some embodiments, provided herein are rAAV viral vectors which do not encode an anti-VEGF Fab or anti-VEGF antibody. In some embodiments, provided herein are rAAV8-based viral vectors which do not encode an anti-VEGF Fab or anti-VEGF antibody. In some embodiments, provided herein are rAAV viral vectors encoding tripeptidyl peptidase 1 (TPP1) protein. In some embodiments, provided herein are rAAV9-based viral vectors encoding TPP1. In some embodiments, provided herein are rAAV viral vectors encoding anti-kallikrein (anti-pKal) protein. In some embodiments, provided herein are rAAV8-based or rAAV9-based viral vectors encoding lanadelumab Fab or full-length antibody. In some embodiments, provided herein are rAAV viral vectors encoding CLN2. In some embodiments, provided herein are rAAV viral vectors encoding CLN3. In some embodiments, provided herein are rAAV viral vectors encoding CLN6. In some embodiments, provided herein are rAAV8-based or rAAV9-based viral vectors encoding CLN2. In some embodiments, provided herein are rAAV8-based or rAAV9-based viral vectors encoding CLN3. In some embodiments, provided herein are rAAV8-based or rAAV9-based viral vectors encoding CLN6.

[0332] In certain embodiments, provided herein are rAAV viral vectors encoding etanercept (an anti-TNF fusion protein). In certain embodiments, provided herein are rAAV viral vectors encoding adalimumab antibody (an anti-TNF antibody) or an antigen-binding fragment thereof. In certain embodiments, provided herein are rAAV viral vectors encoding a full-length anti-C3 or anti-C5 antibody, an anti-C3 or anti-C5 Fab, a complement factor H (CFH), or a complement factor H-like (CFHL-1) protein. In certain embodiments, provided herein are rAAV viral vectors encoding eculizumab, ravulizumab, tesidolumab, crovalimab, NGM621 or BB5.1 antibody, or an antigen-binding fragment thereof.

[0333] In certain embodiments, the therapeutic product (e.g., transgene) is: (1) Palmitoyl-Protein Thioesterase 1 (PPT1); (2) Tripeptidyl-Peptidase 1 (TPP1); (3) Battenin (CLN3); and (4) CLN6 Transmembrane ER Protein (CLN6).

[0334] In certain embodiments, the disclosure provides a pharmaceutical composition comprising recombinant AAV encoding a transgene. In some embodiments, provided herein are rAAV viral vectors encoding tripeptidyl peptidase 1 (TPP1) protein. In some embodiments, provided herein are rAAV9-based viral vectors encoding TPPL. In some embodiments, provided herein are rAAV viral vectors encoding anti-kallikrein (anti-pKal) protein, such as lanadelumab. In some embodiments, provided herein are rAAV8-based or rAAV9-based viral vectors encoding lanadelumab Fab or full-length antibody. In certain embodiments, the rAAV vector is an AAV vector encoding an antibody transgene disclosed in PCT International Application No. PCT / US2020 / 029802 (PCT International Publication No. WO2020219868A1, published Oct. 29, 2020), which is incorporated herein by reference in its entirety.

[0335] In some embodiments, provided herein are rAAV viral vectors encoding CLN2. In some embodiments, provided herein are rAAV viral vectors encoding CLN3. In some embodiments, provided herein are rAAV viral vectors encoding CLN6. In some embodiments, provided herein are rAAV8-based or rAAV9-based viral vectors encoding CLN2. In some embodiments, provided herein are rAAV8-based or rAAV9-based viral vectors encoding CLN3. In some embodiments, provided herein are rAAV8-based or rAAV9-based viral vectors encoding CLN6.

[0336] In certain embodiments, the vectors provided herein can be used for (1) the pathology of the eye associated with Batten-CLN1 and the therapeutic product is Palmitoyl-Protein Thioesterase 1 (PPT1); (2) the pathology of the eye associated with Batten-CLN2 and the therapeutic product is Tripeptidyl-Peptidase 1 (TPP1); (3) the pathology of the eye associated with Batten-CLN3 and the therapeutic product is Battenin (CLN3); (4) the pathology of the eye associated with Batten-CLN6 and the therapeutic product is CLN6 Transmembrane ER Protein (CLN6); (5) the pathology of the eye associated with Batten-CLN7 and the therapeutic product is Major Facilitator Superfamily Domain Containing 8 (MVFSD8); and (6) the pathology of the eye associated with Batten-CLN1 and the therapeutic product is Palmitoyl-Protein Thioesterase 1 (PPT1).TABLE 1Exemplary sequencesSEQ IDNO:DescriptionSequence5VEGF-A signalMNFLLSWVHW SLALLLYLHH AKWSQApeptide6Fibulin-1 signalMERAAPSRRV PLPLLLLGGL ALLAAGVDApeptide7VitronectinMAPLRPLLIL ALLAWVALAsignal peptide8ComplementMRLLAKIICLMLWAICVAFactor H signalpeptide9Opticin signalMRLLAFLSLL ALVLQETGTpeptide22Albumin signalMKWVTFISLLFLFSSAYSpeptide23ChymotrypsinogMAFLWLLSCWALLGTTFGen signal peptide24Interleukin-2MYRMQLLSCIALILALVTNSsignal peptide25Trypsinogen-2MNLLLILTFVAAAVAsignal peptide26F2A siteLLNFDLLKLAGDVESNPGP27T2A site(GSG)EGRGSLLTCGDVEENPGP28P2A site(GSG)ATNFSLLKQAGDVEENPGP29E2A site(GSG)QCTNYALLKLAGDVESNPGP30F2A site(GSG)VKQTLNFDLLKLAGDVESNPGP31Furin linkerRKRR32Furin linkerRRRR33Furin linkerRRKR34Furin linkerRKKR35Furin linkerR-X-K / R-R36Furin linkerRXKR37Furin linkerRXRR41AAV1MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL42AAV2MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL43AAV3-3MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGYKYLGPGNGLDKGEPVNEADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAAKTAPGKKGAVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVRGVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTGTVNHQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL44AAV4-4MTDGYLPDWLEDNLSEGVREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQQRLQGDTSFGGNLGRAVFQAKKRVLEPLGLVEQAGETAPGKKRPLIESPQQPDSSTGIGKKGKQPAKKKLVFEDETGAGDGPPEGSTSGAMSDDSEMRAAAGGAAVEGGQGADGVGNASGDWHCDSTWSEGHVTTTSTRTWVLPTYNNHLYKRLGESLQSNTYNGFSTPWGYFDFNRFHCHFSPRDWQRLINNNWGMRPKAMRVKIFNIQVKEVTTSNGETTVANNLTSTVQIFADSSYELPYVMDAGQEGSLPPFPNDVFMVPQYGYCGLVTGNTSQQQTDRNAFYCLEYFPSQMLRTGNNFEITYSFEKVPFHSMYAHSQSLDRLMNPLIDQYLWGLQSTTTGTTLNAGTATTNFTKLRPTNFSNFKKNWLPGPSIKQQGFSKTANQNYKIPATGSDSLIKYETHSTLDGRWSALTPGPPMATAGPADSKFSNSQLIFAGPKQNGNTATVPGTLIFTSEEELAATNATDTDMWGNLPGGDQSNSNLPTVDRLTALGAVPGMVWQNRDIYYQGPIWAKIPHTDGHFHPSPLIGGFGLKHPPPQIFIKNTPVPANPATTFSSTPVNSFITQYSTGQVSVQIDWEIQKERSKRWNPEVQFTSNYGQQNSLLWAPDAAGKYTEPRAIGTRYLTHHL45AAV5MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL46AAV6MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPS...

Claims

1. A pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject, wherein the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, and wherein the pharmaceutical composition has an amount of viral vector aggregation such that when administered to an eye of a pig:a. the clearance time of the pharmaceutical composition is between about 5 days and about 15 days; andb. the thickness of the SCS at the site of injection is between about 400 μm and about 800 μm at a time within one hour of administration; andc. the circumferential spread of the pharmaceutical composition from the site of injection is about one-eighth or less of a surface of the choroid at a time within about one hour of administration.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises an ionic strength of at most about 200 mM prior to suprachoroidal administration.

3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises at least about 3% aggregated recombinant AAV prior to suprachoroidal administration.

4. The pharmaceutical composition of any one of claims 1-3, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is equal to or greater than the clearance time after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition.

5. The pharmaceutical composition of any one of claims 1-3, wherein a circumferential spread after suprachoroidal administration of the pharmaceutical composition is smaller as compared to a circumferential spread after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition.

6. The pharmaceutical composition of any one of claims 1-3, wherein a thickness at a site of injection after suprachoroidal administration of the pharmaceutical composition is equal to or higher as compared to a thickness at a site of injection after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition.

7. The pharmaceutical composition of any one of claims 1-3, wherein an expression level of the transgene is detected in the eye for a longer period of time after suprachoroidal administration of the pharmaceutical composition as compared to a period of time that an expression level of the transgene is detected in the eye after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition.

8. The pharmaceutical composition of any one of claims 1-3, wherein the concentration of the transgene product in the eye after suprachoroidal administration of the pharmaceutical composition is equal to or higher as compared to the concentration of the transgene product in the eye after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition;optionally wherein the concentration of the transgene product in the back of the eye (e.g., retina) after suprachoroidal administration of the pharmaceutical composition is equal to or higher as compared to the concentration of the transgene product in the back of the eye after suprachoroidal administration of a reference pharmaceutical composition, and / or the concentration of the transgene product in the outer layer of the eye (e.g., sclera) after suprachoroidal administration of the pharmaceutical composition is lower than the concentration of the transgene product in the outer layer of the eye after suprachoroidal administration of a reference pharmaceutical composition.

9. The pharmaceutical composition of any one of claims 1-3, wherein the rate of transduction at a site of injection after suprachoroidal administration is equal to or higher as compared to the rate of transduction at a site of injection after suprachoroidal administration of a reference pharmaceutical composition, wherein the reference pharmaceutical composition comprises the recombinant AAV comprising the expression cassette encoding the transgene, wherein an amount of the recombinant AAV genome copies is the same when the pharmaceutical composition or the reference pharmaceutical composition is administered to the suprachoroidal space, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the reference pharmaceutical composition.

10. The pharmaceutical composition of any one of claims 2-9, wherein the transgene is not an anti-human vascular endothelial growth factor (anti-VEGF) antibody.

11. The pharmaceutical composition of any one of claims 2-10, wherein the recombinant AAV comprises components from one or more adeno-associated virus serotypes selected from the group consisting of AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, rAAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

12. The pharmaceutical composition of any one of claims 1-11, wherein the recombinant AAV is AAV8.

13. The pharmaceutical composition of any one of claims 1-11, wherein the recombinant AAV is AAV9.

14. The pharmaceutical composition of any one of claims 1-13, wherein the pharmaceutical composition has an ionic strength of about or at most about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM.

15. The pharmaceutical composition of any one of claims 1-14, wherein the pharmaceutical composition comprises at least about or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% aggregated recombinant AAV.

16. The pharmaceutical composition of any one of claims 1-15, wherein the pharmaceutical composition has an ionic strength of about or of at most about 40 mM.

17. The pharmaceutical composition of any one of claims 1-15, wherein the pharmaceutical composition has an ionic strength of about or of at most about 135 mM.

18. The pharmaceutical composition of any one of claims 1-15, wherein the pharmaceutical composition has an ionic strength of about or of at most about 20 mM.

19. The pharmaceutical composition of any one of claims 1-18, wherein the pharmaceutical composition has an average recombinant AAV diameter of about or at least about: 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, or about or at least about 100 nm as measured by dynamic light scattering.

20. The pharmaceutical composition of any one of claims 4-19, wherein the pharmaceutical composition has an average recombinant AAV diameter that is at least 2 times higher, at least 3 times higher, at least 4 times higher, at least 5 times higher, at least 6 times higher, at least 7 times higher, at least 8 times higher, at least 9 times higher, at least 10 times higher, at least 15 times higher, at least 20 times higher, at least 50 times higher, at least 100 times higher, at least 5% higher, at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40%, at least 45% higher, at least 50% higher, at least 55% higher, at least 60% higher, at least 65% higher, at least 70% higher, at least 75% higher, at least 80% higher, at least 85% higher, at least 90% higher, at least 95% higher, at least 100% higher, at least 150% higher, or at least 200% higher, at least 250% higher, or at least 300%, at least 400% higher, or at least 500% higher than an average recombinant AAV diameter in the reference pharmaceutical composition.

21. The pharmaceutical composition of any one of claims 5 and 10-20, wherein the circumferential spread after suprachoroidal administration of the pharmaceutical composition is smaller by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%.

22. The pharmaceutical composition of any one of claims 4 and 10-21, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is greater by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or at least 500%.

23. The pharmaceutical composition of any one of claims 1-22, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is of about 6 days to about 15 days, about 7 days to about 15 days, about 8 days to about 15 days, about 9 days to about 15 days, about 10 days to about 15 days, about 11 days to about 15 days, about 12 days to about 15 days, about 13 days to about 15 days, about 14 days to about 15 days, about 5 days to about 14 days, about 5 days to about 13 days, about 5 days to about 12 days, about 5 days to about 11 days, about 5 days to about 10 days, about 5 days to about 9 days, about 5 days to about 8 days, about 5 days to about 7 days, or about 5 days to about 6 days.

24. The pharmaceutical composition of any one of claims 1-23, wherein the clearance time after suprachoroidal administration of the pharmaceutical composition is not prior to about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

25. The pharmaceutical composition of any one of claims 4-24, wherein the clearance time after suprachoroidal administration of the reference pharmaceutical composition is of at most about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

26. The pharmaceutical composition of any one of claims 1-25, wherein the clearance time is from the SCS or from the eye.

27. The pharmaceutical composition of any one of claims 1-26, wherein the clearance time is the time required for the pharmaceutical composition and / or the recombinant adeno-associated virus (AAV) vector to not be detectable in the SCS by any standard method.

28. The pharmaceutical composition of any one of claims 1-26, wherein the clearance time when the pharmaceutical composition and / or the recombinant adeno-associated virus (AAV) vector is present in the SCS in an amount that is at most about 2% or at most about 5% of the amount detectable by any standard method.

29. The pharmaceutical composition of any one of claims 1-26, wherein the clearance time is the amount of time required following injection for the thickness at the site of injection to decrease to about 500 nm or less, about 200 nm or less, about 100 nm or less, about 50 nm or less, about 25 nm or less, about 10 nm or less, or is undetectable.

30. The pharmaceutical composition of any one of claims 1-26, wherein the clearance time is the amount of time required following injection for the pharmaceutical composition to spread circumferentially from the site of injection to cover about one-sixteenth or more, about one-eighth or more, about one-fourth or more, about one-half or more, about three-fourths or more, or all of the circumference of the choroid of the eye.

31. The pharmaceutical composition of any one of claims 6 and 10-30, wherein the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is higher by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%.

32. The pharmaceutical composition of any one of claims 1-31, wherein the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is about 500 μm to about 3.0 mm, 750 μm to about 2.8 mm, about 750 μm to about 2.5 mm, about 750 μm to about 2 mm, or about 1 mm to about 2 mm.

33. The pharmaceutical composition of any one of claims 1-32, wherein the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition is of at least about 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, or 700 μm, 800 μm, 900 μm, 1000 μm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm.

34. The pharmaceutical composition of any one of claims 6 and 10-33, wherein the thickness at the site of injection after suprachoroidal administration of the reference pharmaceutical composition is of at most about 1 nm, 5 nm, 10 nm, 25 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm.

35. The pharmaceutical composition of any one of claims 1-34, wherein the thickness of the SCS at the site of injection is about 400 μm to about 700 μm, about 400 μm to about 600 μm, about 400 μm to about 500 μm, about 500 μm to about 800 μm, about 600 μm to about 800 μm, 700 μm to about 800 μm at a time within one hour of administration.

36. The pharmaceutical composition of claim 35, wherein the time within one hour of administration is within about 5 minutes of administration, within about 10 minutes of administration, within about 15 minutes of administration, within about 20 minutes of administration, within about 30 minutes of administration, within about 45 minutes of administration, or within about 60 minutes of administration.

37. The pharmaceutical composition of any one of claims 1-36, wherein the thickness at the site of injection after suprachoroidal administration of the pharmaceutical composition persists for at least two hours, at least three hours, at least four hours, at least five hours, at least six hours, at least seven hours, at least eight hours, at least ten hours, at least twelve hours, at least eighteen hours, at least twenty-four hours, at least two days, at least three days, at least five days, at least ten days, at least twenty-one days, at least one month, at least six weeks, at least two months, at least three months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least one year, at least three years, or at least five years.

38. The pharmaceutical composition of any one of claims 1-36, wherein the circumferential spread of the pharmaceutical composition from the site of injection is about one-eighth or less of a surface of the choroid at a time within about 5 minutes of administration, within about 10 minutes of administration, within about 15 minutes of administration, within about 20 minutes of administration, within about 30 minutes of administration, within about 45 minutes of administration, or within about 60 minutes of administration.

39. The pharmaceutical composition of claim 38, wherein the circumferential spread from the site of injection is about one-sixteenth or less of a surface of the choroid.

40. The pharmaceutical composition of any one of claims 8 and 10-39, wherein the concentration of the transgene in the eye after suprachoroidal administration of the pharmaceutical composition is higher by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%.

41. The pharmaceutical composition of any one of claims 7 and 10-40, wherein the longer period of time after suprachoroidal administration of the pharmaceutical composition is longer by at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days.

42. The pharmaceutical composition of any one of claims 1-41, wherein the transgene is detected in the eye after suprachoroidal administration of the pharmaceutical composition for at least about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days.

43. The pharmaceutical composition of any one of claims 4-42, wherein the transgene is detected in the eye after suprachoroidal administration of the reference pharmaceutical composition for at most about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 23 days, 25 days, 27 days, 30 days, 35 days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, or 100 days, 120 days, 140 days, 160 days, 180 days, 200 days, 220 days, 240 days, 260 days, 280 days, 300 days, 320 days, 340 days, 360 days, 380 days, or 400 days after.

44. The pharmaceutical composition of any one of claims 9 and 10-43, wherein the rate of transduction at the site of injection after suprachoroidal administration of the pharmaceutical composition is higher by at least about 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 100 times, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, at least 250%, or at least 300%, at least 400%, or by at least 500%.

45. The pharmaceutical composition of any one of claims 2-44, wherein the recombinant AAV stability in the pharmaceutical composition is at least about 50% of the recombinant AAV stability in the reference pharmaceutical composition.

46. The pharmaceutical composition of claim 45, wherein the recombinant AAV stability is determined by infectivity of the recombinant AAV.

47. The pharmaceutical composition of claim 45, wherein the recombinant AAV stability is determined by a level of free DNA released by the recombinant AAV.

48. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition comprises at least about 50% more, about 25% more, about 15% more, about 10% more, about 5% more, about 4% more, about 3% more, about 2% more, about 1% more, about 0% more, about 1% less, about 2% less, about 5% less, about 7% less, about 10% less, about 2 times more, about 3 times more, about 2 times less, about 3 times less free DNA as compared to a level of free DNA in the reference pharmaceutical composition.

49. The pharmaceutical composition of claim 46, wherein the recombinant AAV in the pharmaceutical composition has an infectivity that is about 50% lower, about the same, or at least about 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30% 35% 40%, 45%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 100 times, or 1000 times higher as compared to the infectivity of the recombinant AAV in the reference pharmaceutical composition.

50. The pharmaceutical composition of any one of claims 1-49, wherein the transgene is a transgene suitable to treat, or otherwise ameliorate, prevent or slow the progression of a disease of interest.

51. The pharmaceutical composition of any one of claims 1-50, wherein the human subject is diagnosed with nAMD (wet AMID), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), or Batten disease.

52. The pharmaceutical composition of any one of claims 1-50, wherein the human subject is diagnosed with glaucoma, non-infectious uveitis, or kallikrein-related disease.

53. The pharmaceutical composition of any one of claims 1-3, 4-9, and 10-52, wherein the AAV encodes Palmitoyl-Protein Thioesterase 1 (PPT1), Tripeptidyl-Peptidase 1 (TPP1), anti-TNF fusion protein, anti-kallikrein antibody or antigen-binding fragment, anti-TNF antibody or antigen-binding fragment, anti-C3 antibody or antigen-binding fragment, or anti-C5 antibody or antigen-binding fragment.

54. The pharmaceutical composition of any one of claims 4-53, wherein the amount of the recombinant AAV genome copies is based on a vector genome concentration.

55. The pharmaceutical composition of any one of claims 4-53, wherein the amount of the recombinant AAV genome copies is based on genome copies per administration.

56. The pharmaceutical composition of any one of claims 4-53, wherein the amount of the recombinant AAV genome copies is based on total genome copies administered to the human subject.

57. The pharmaceutical composition of claim 55, wherein the genome copies per administration is the genome copies of the recombinant AAV per suprachoroidal administration.

58. The pharmaceutical composition of claim 56, wherein the total genome copies administered is the total genome copies of the recombinant AAV administered suprachoroidally.

59. The pharmaceutical composition of claim 54, wherein the vector genome concentration (VGC) is of about 3×109 GC / mL, about 1×1010 GC / mL, about 1.2×1010 GC / mL, about 1.6×1010 GC / mL, about 4×1010 GC / mL, about 6×1010 GC / mL, about 2×1011 GC / mL, about 2.4×1011 GC / mL, about 2.5×1011 GC / mL, about 3×1011 GC / mL, about 6.2×1011 GC / mL, about 1×1012 GC / mL, about 2.5×1012 GC / mL, about 3×1012 GC / mL, about 5×1012 GC / mL, about 6×1012 GC / mL, about 1.5×1013 GC / mL, about 2×1013 GC / mL, or about 3×1013 GC / mL.

60. The pharmaceutical composition of any one of claims 56 and 58, wherein the total number of genome copies administered is about 6.0×1010 genome copies, about 1.6×1011 genome copies, about 2.5×1011 genome copies, about 3×1011 genome copies, about 5.0×1011 genome copies, about 6×1011 genome copies, about 3×1012 genome copies, about 1.0×1012 genome copies, about 1.5×1012 genome copies, about 2.5×1012 genome copies, or about 3.0×1013 genome copies.

61. The pharmaceutical composition of any one of claims 55 and 57, wherein the total number of genome copies per administration is about 6.0×1010 genome copies, about 1.6×1011 genome copies, about 2.5×1011 genome copies, about 3×1011 genome copies, about 5.0×1011 genome copies, about 6×1011 genome copies, about 3×1012 genome copies, about 1.0×1012 genome copies, about 1.5×1012 genome copies, about 2.5×1012 genome copies, or about 3.0×1013 genome copies.

62. The pharmaceutical composition of any one of claims 2-61, wherein the pharmaceutical composition is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, twenty five times, or thirty times.

63. The pharmaceutical composition of any one of claims 4-62, wherein the reference pharmaceutical composition is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, twenty-five times, or thirty times.

64. The pharmaceutical composition of any one of claims 2-63, wherein the pharmaceutical composition is administered once in one day, twice in one day, three times in one day, four times in one day, five times in one day, six times in one day, or seven times in one day.

65. The pharmaceutical composition of any one of claims 4-63, wherein the reference pharmaceutical composition is administered once in one day, twice in one day, three times in one day, four times in one day, five times in one day, six times in one day, or seven times in one day.

66. The pharmaceutical composition of any one of claims 2-65, wherein the reference pharmaceutical composition comprises DPBS and sucrose.

67. A method of preparing a pharmaceutical composition comprising:a. preparing a composition comprising phosphate-buffered saline, sucrose, and a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene; andb. admixing a solution comprising phosphate-buffered saline and sucrose to the composition,wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the composition.

68. A method of preparing a pharmaceutical composition comprising admixing a solution comprising phosphate-buffered saline and sucrose to a composition, wherein the composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, and wherein the pharmaceutical composition has lower ionic strength and / or a higher level of aggregated recombinant AAV than the composition.

69. A kit comprising:a. a composition comprising a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene; andb. a solution comprising phosphate-buffered saline and sucrose.

70. The kit of claim 69, wherein the kit further comprises instructions for admixing the composition with the solution.

71. The method of claim 68 or the kit of claim 69, wherein the composition comprises a phosphate-buffered saline and sucrose.

72. The method of any one of claims 67 and 71, or the kit of any one of claims 69 and 71, wherein the composition comprises 4% sucrose.

73. The method of any one of claims 67, 68, 71 and 72, or the kit of any one of claims 69-72, wherein the solution comprises 10% sucrose.

74. The method of any one of claims 67, 68, and 71-73, or the pharmaceutical composition of any one of claims 1-66, wherein the pharmaceutical composition has an ionic strength of about or of at most about 135 mM.

75. The method of any one of claims 67, 68, and 71-74, or the pharmaceutical composition of any one of claims 1-66, wherein the pharmaceutical composition has an ionic strength of about or of at most about 40 mM.

76. The method of any one of claims 67, 68, and 71-75, or the pharmaceutical composition of any one of claims 1-66, wherein the pharmaceutical composition has an ionic strength of about or of at most about 20 mM.

77. The method of any one of claims 67, 68, and 71-76, or the pharmaceutical composition of any one of claims 1-66, wherein the pharmaceutical composition has substantially the same tonicity or osmolality as the composition.

78. The method of any one of claims 67, 68, and 71-77, or the pharmaceutical composition of any one of claims 1-66, wherein at least some of the aggregated recombinant AAV in the pharmaceutical composition disaggregate after the pharmaceutical composition is administered to the suprachoroidal space of an eye of a human subject.

79. The method of any one of claims 67, 68, and 71-78, or the pharmaceutical composition of any one of claims 1-66, wherein aggregation of the recombinant AAV is reversed to unaggregated AAV or to monomers upon suprachoroidal administration of the pharmaceutical composition.

80. The method of any one of claims 67, 68, and 71-79, or the kit of any one of claims 69-72, wherein the composition comprises potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and optionally a surfactant.

81. The method of any one of claims 67, 68, and 71-80, or the kit of any one of claims 69-72 and 80, wherein the composition comprises modified Dulbecco's phosphate-buffered saline solution, and optionally a surfactant.

82. The method of any one of claims 67, 68, and 71-81, or the kit of any one of claims 69-72 and 80-81, wherein the composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, and a surfactant.

83. The method of any one of claims 67, 68, and 71-82, or the kit of any one of claims 69-72 and 80-82, wherein the solution comprises a phosphate-buffered sodium chloride and sucrose.

84. The kit of claim 70, wherein the instructions comprise instructions on admixing the solution with the composition to obtain a pharmaceutical composition.

85. The method of any one of claims 67, 68, and 71-83, or the kit of claim 84, wherein the admixing the solution with the composition dilutes the composition by about or at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold.

86. The method of any one of claims 67, 68, 71-83 and 85, or the kit of any one of claims 84-85, wherein the admixing the solution with the composition occurs on the same day that the pharmaceutical composition is administered to the suprachoroidal space of an eye of a human subject.

87. The method of any one of claims 67, 68, 71-83 and 85-86, or the kit of any one of claims 84-86, wherein the admixing the solution with the composition occurs within 24 hours of the pharmaceutical composition being administered to the suprachoroidal space of an eye of a human subject.

88. The method of any one of claims 67, 68, 71-83 and 85-87, or the kit of any one of claims 84-87, or the pharmaceutical composition of any one of claims 2-66 and 74-79, wherein the pharmaceutical composition is stored prior to administration to a human subject.

89. The method of any one of claims 67, 68, 71-83 and 85-88, or the kit of any one of claims 84-88, or the pharmaceutical composition of any one of claims 2-66, 74-79, and 88, wherein the pharmaceutical composition is stored at about room temperature, 20° C., 4° C., or −80° C.

90. The method of any one of claims 67, 68, 71-83 and 85-89, or the kit of any one of claims 84-89, or the pharmaceutical composition of any one of claims 2-66, 74-79, and 88-89, wherein the pharmaceutical composition comprises about 1.0×1012 to about 3.0×1012 genome copies of the recombinant AAV.

91. The method of any one of claims 67, 68, 71-83 and 85-90, or the kit of any one of claims 84-90, wherein the recombinant AAV comprises components from one or more adeno-associated virus serotypes selected from AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, rAAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

92. The method of any one of claims 67, 68, 71-83 and 85-91, or the kit of any one of claims 84-91, or the pharmaceutical composition of any one of claims 2-66, 74-79, and 88-90, wherein the recombinant AAV comprises components from AAV8 and the pharmaceutical composition has an ionic strength between about 30 mM to about 60 mM.

93. The method of any one of claims 67, 68, 71-83 and 85-91, or the kit of any one of claims 84-91, or the pharmaceutical composition of any one of claims 2-66, 74-79, and 88-90, wherein the recombinant AAV comprises components from AAV9 and the pharmaceutical composition has an ionic strength between about 15 mM to about 30 mM.

94. The method of any one of claims 67, 68, 71-83 and 85-91, or the kit of any one of claims 84-91, or the pharmaceutical composition of any one of claims 1-66, 74-79, and 88-90, wherein the recombinant AAV comprises components from AAV2 and the pharmaceutical composition has an ionic strength between about 100 mM to about 200 mM.

95. The pharmaceutical composition of any one of claims 1-66, 74-79, and 88-94, wherein the pharmaceutical composition comprises modified Dulbecco's phosphate-buffered saline solution, and optionally a surfactant.

96. The pharmaceutical composition of any one of claims 1-66, 74-79, and 88-95, wherein the pharmaceutical composition comprises potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and optionally a surfactant.

97. The pharmaceutical composition of any one of claims 1-66, 74-79, and 88-96, wherein the pharmaceutical composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, and optionally a surfactant.

98. The pharmaceutical composition of any one of claims 1-66, 74-79, and 88-96, wherein the pharmaceutical composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, 0.001% (0.01 mg / mL) poloxamer 188, pH 7.4.

99. A pharmaceutical composition suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject, wherein the pharmaceutical composition comprises a recombinant adeno-associated virus (AAV) vector comprising an expression cassette encoding a transgene, wherein the pharmaceutical composition comprises 0.2 mg / mL potassium chloride, 0.2 mg / mL potassium phosphate monobasic, 5.84 mg / mL sodium chloride, 1.15 mg / mL sodium phosphate dibasic anhydrous, 40.0 mg / mL (4% w / v) sucrose, 0.001% (0.01 mg / mL) poloxamer 188, pH 7.4.

100. The pharmaceutical composition of any one of claims 1-66, 74-79, and 88-99, wherein the pharmaceutical composition comprises a lower amount of AAV empty capsids as compared to a reference pharmaceutical composition.

101. The pharmaceutical composition of claim 100, wherein the amount of the AAV empty capsids in the pharmaceutical composition is lower by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the amount of the AAV empty capsids in the reference pharmaceutical composition.

102. The pharmaceutical composition of any one of claims 1-66, 74-79, and 88-101, wherein the pharmaceutical composition comprises an ionic strength of about or at most about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, or 60 mM.

103. The pharmaceutical composition of any one of claims 100-102, wherein the reference pharmaceutical composition comprises an ionic strength of more than about 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, or more than about 200 mM.

104. A method of reducing or eliminating AAV empty capsids in a pharmaceutical composition, the method comprising:a. introducing a solution comprising an ionic strength of at most about 60 mM to a formulation comprising a mixture of a recombinant adeno-associated virus (AAV) vector and AAV empty capsids; andb. removing at least some of the AAV empty capsids from the formulation, wherein the formulation after step b is prepared into a pharmaceutical composition comprising the recombinant adeno-associated virus (AAV) vector, wherein the recombinant AAV vector comprises an expression cassette encoding a transgene, and wherein the pharmaceutical composition is suitable for administration to the suprachoroidal space (SCS) of an eye of a human subject.

105. A method of reducing or eliminating AAV empty particles in a population of AAV particles, wherein the population of AAV particles comprises empty AAV particles and AAV particles comprising an expression cassette encoding a transgene, and wherein the method comprises:a. incubating the population of AAV particles in a solution at an ionic strength of at most about 60 mM, thereby creating aggregates of the AAV particles comprising the expression cassette encoding the transgene; andb. removing at least a portion of the empty AAV particles from the population of AAV particles.

106. The method of claim 104, wherein the method further comprises introducing another solution comprising an ionic strength of at least about 80 mM to the formulation after step b.

107. The method of claim 105, wherein the method further comprises incubating the population of AAV particles after step b with another solution comprising an ionic strength of at least about 80 mM.

108. The method of claim 104 or 106, wherein the amount of the AAV empty capsids in the formulation is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99%, or 100% after step b. as compared to the amount of the AAV empty capsids in the formulation prior to step a.

109. The method of claim 105 or 107, wherein the amount of the empty AAV particles in the population of AAV particles is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99%, or 100% after step b. as compared to the amount of the empty AAV particles in the population of AAV particles prior to step a.

110. The method of any one of claims 104-109, wherein the solution comprises an ionic strength of about 30 to about 50 mM.

111. The method of any one of claims 104-109, wherein the solution comprises an ionic strength of about 15 to 50 mM.

112. The method of any one of claims 104-109, wherein the solution comprises an ionic strength of at most about 50 mM.

113. The method of any one of claims 104-109, wherein the another solution comprises an ionic strength of about or at least about 150 mM.

114. A pharmaceutical composition produced by the method of any one of claims 104, 106, and 108-113.

115. A pharmaceutical composition comprising the population of AAV particles obtained after step b of the method of any one of claims 105 and 107-113.

116. The method or the kit of claim 81 or 82, wherein the solution comprises 10% Sucrose, 2.70 mM potassium chloride, 8.10 mM sodium phosphate dibasic anhydrous, 1.47 mM potassium phosphate monobasic, 292 mM sucrose, 0.001% (0.01 mg / mL) poloxamer 188, pH 7.4, and wherein the composition and solution are admixed at a composition to solution ratio of 1 to 9.

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